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Human Molecular Genetics Advance Access originally published online on March 2, 2007
Human Molecular Genetics 2007 16(7):753-762; doi:10.1093/hmg/ddm006
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Lafora disease proteins malin and laforin are recruited to aggresomes in response to proteasomal impairment

Shuchi Mittal1, Deepti Dubey1, Kazuhiro Yamakawa2 and Subramaniam Ganesh1,*

1 Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, 208016, India and 2 Laboratory for Neurogenetics, RIKEN Brain Sciences Institute, Wako-shi, 351-0198, Japan

* To whom Correspondence should be addressed. Tel: +91 5122594040; Fax: +91 5122594010; Email: sganesh{at}iitk.ac.in

Received December 14, 2006; Revised December 14, 2006; Accepted January 29, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Lafora disease (LD), an autosomal recessive neurodegenerative disorder, is characterized by the presence of cytoplasmic polyglucosan inclusions known as Lafora bodies in several tissues including the brain. Laforin, a protein phosphatase, and malin, an ubiquitin ligase, are two of the proteins that are known to be defective in LD. Malin interacts with laforin and promotes its polyubiquitination and degradation. Here we show that malin and laforin co-localize in endoplasmic reticulum (ER) and that they form centrosomal aggregates when treated with proteasomal inhibitors in both neuronal and non-neuronal cells. Laforin/malin aggregates co-localize with {gamma}-tubulin and cause redistribution of {alpha}-tubulin. These aggregates are also immunoreactive to ubiquitin, ubiquitin-conjugating enzyme, ER chaperone and proteasome subunits, demonstrating their aggresome-like properties. Furthermore, we show that the centrosomal aggregation of laforin and malin is dependent on the functional microtubule network. Laforin and malin form aggresome when expressed together or otherwise, suggesting that the two proteins are recruited to the centrosome independent of each other. Taken together, our results suggest that the centrosomal accumulation of malin, possibly with the help of laforin, may enhance the ubiquitination of its substrates and facilitate their efficient degradation by proteasome. Defects in malin or laforin may thus lead to increased levels of misfolded and/or target proteins, which may eventually affect the physiological processes of the neuron. Thus, defects in protein degradation and clearance are likely to be the primary trigger in the physiopathology of LD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Lafora disease (LD), which presents in teenage years, is one of the five forms of inherited progressive myoclonus epilepsies (1,2). LD patients display symptoms of stimulus-sensitive grand mal, tonic–clonic, absence, visual and myoclonic seizures (1). Rapidly progressive dementia, psychosis, cerebellar ataxia, dysarthria, mutism, muscle wasting and respiratory failure lead to death within 10 years of the onset (1). One of the characteristic features of LD is the presence of Lafora polyglucosan bodies. Histochemical studies have suggested that the inclusions are a mixture of acid mucopolysaccharides and glycolproteins (3). Lafora bodies stain positive for anti-ubiquitin and anti-advanced glycation end products antibodies, suggesting that these inclusions are not only rich in glucose but also contain higher levels of glycosaminoglycans (4,5). LD is caused by mutations in the EPM2A gene encoding laforin, a dual-specificity phosphatase, or in the NHLRC1 gene encoding malin, an ubiquitin ligase (6–10). Laforin and malin, to be collectively referred to as LD proteins, regulate functions of, as yet unidentified, cellular targets by their post-translational modifications (2). Malin is also known to interact with laforin and promote the polyubiquitination and degradation of laforin in vitro and in cultured cells (10,11). However, the cellular function of malin could not be limited to laforin's degradation, as defects in this process will not explain locus heterogeneity observed in LD, an autosomal recessive disorder (2). For example, defects in malin will lead to increased levels of laforin and, on the contrary, null mutations in EPM2A will result in absence of laforin; however, recessive mutations in either EPM2A or NHLRC1 result in LD. Thus, laforin and malin together are likely to regulate critical processes involved in the pathogenesis of LD (2).

The ubiquitin-proteasome system (UPS) is the major non-lysosomal pathway for intracellular protein degradation (12–14). UPS relies on transfer of ubiquitin molecules to the target protein through three enzymatic steps, with the key steps of substrate selection and the transfer of ubiquitin being delegated to an enzyme called the E3 or the ubiquitin-protein ligase (15). The presence of ubiquitin-positive protein aggregates in major neurodegenerative disorders suggests that dysfunctions in UPS, either due to its malfunction in components of UPS or overload of the system due to aggregation of unfolded/mutant proteins, is likely to promote neurodegenerative process (13,14,16,17). The discovery that malin is an ubiquitin-protein ligase further highlights the importance of UPS in neurological disorders. Malin is only the second E3 enzyme known to have a direct role in a neurodegenerative disorder. The other one is Parkin, mutated in the majority of cases with juvenile-onset Parkinsonism (18). Both malin and Parkin are RING finger ubiquitin ligases and cause disease phenotype in autosomal recessive conditions (9,18,19). Several studies have shown that inhibition of proteasomal activity causes Parkin to aggregate, which co-localizes with ubiquitin and proteasome (20–23). In the present report, we show that, similar to Parkin, proteasomal inhibition leads to the accumulation of malin and laforin as perinuclear aggregates, and these structures are aggresomes. On the basis of our observations, we suggest that both malin and laforin are involved in UPS and defects in protein degradation and clearance could underlie the pathological process of LD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Proteasomal inhibition induces formation of inclusion in cells expressing malin or laforin
In order to investigate the subcellular localization pattern of malin, we generated several expression constructs (see Materials and Methods). These include malin with N-terminal green fluorescent protein (GFP), or the FLAG-tag, and C-terminal Myc-tag and a malin construct without any tag. Fluorescence imaging for malin expression revealed its localization in both cytoplasm and nucleus, with predominant staining coming from the perinuclear region (Fig. 1A and Supplementary Material, Fig. S1). The subcellular localization pattern observed for malin was consistent regardless of the cell line used (COS-7, SH-SY5Y or HEK293) or the fusion tags used (Supplementary Material, Fig. S1A and B). Identical pattern of localization was observed when malin was expressed without any tags and detected using a polyclonal antibody specific to malin, suggesting that addition of tags to malin does not alter its subcellular localization (Supplementary Material, Fig. S1D). Therefore, subsequent studies were done with cells expressing either Myc- or GFP-tagged malin, as it allowed flexibility in antibody combination for double staining. As reported earlier (8), laforin showed reticular cytoplasmic localization (Fig. 1A).


Figure 1
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  		Figure 1. Proteasomal inhibition induces laforin and malin to form perinuclear aggregates: GFP-tagged malin or laforin was transiently expressed in COS-7 (A) or SH-SY5Y cells (B). Although laforin's expression was predominantly cytoplasmic, malin was present both in nucleus and cytoplasm (A; also see Supplementary Material, Fig. S1A). Addition of the proteasomal inhibitor MG132 led to the perinuclear aggregation of laforin and malin when individually expressed in COS-7 or SH-SY5Y cells as indicated (A and B). Cells were counterstained with DAPI (blue color) to visualize nuclei/nucleus. MG132 treatment did not alter the subcellular localization of GFP-tagged endobrevin, a membrane protein, when overexpressed under identical conditions in COS-7 cells (C). Endobrevin showed typical punctuate endosomal staining (C) (25). As shown earlier (24), the cellular distribution of GFP–CFTR was significantly altered when the cells were treated with MG132, developing the characteristic perinuclear aggresome (C). Proteasome blockade increased the cellular levels of malin and laforin (D). COS-7 cells transiently expressing Myc-tagged malin (~50 kDa) or laforin (~47 kDa) were treated with 5 µM MG132 or the vehicle (DMSO) (–) for 24 h as indicated. Ten micrograms of each total cell lysate was separated by SDS–PAGE and immunoblotted with anti-Myc antibody (D). Closed arrow indicates the elevated levels of malin or laforin in cells treated with MG132. Open arrow depicts reactivity of an antibody for ß-actin protein, serving as loading control. Aggregation of malin/laforin is a specific response to the proteasomal stress (E–G); COS-7 cells, transiently expressing GFP-malin (E) or GFP-laforin (F), were treated with 20 µM MG132, 10 µg/ml lactacystin, 300 mM sorbitol, 10 µg/ml tunicamycin, 100 µg/ml MMS or 400 µM hydrogen peroxide (H2O2), as mentioned in the Materials and Methods section. Percent transfected cells showing normal distribution for laforin/malin or aggregates (A) were scored and plotted. For both malin (E) and laforin (F), the value of each treatment represents the mean average of three independent assays, with a minimum of 300 transfected cells scored for each assay. Error bars indicate the standard error of the mean. A difference in the number of cells having laforin or malin aggregates between the treated groups and the time-matched untreated group was calculated for statistical significance by paired t-test. A P-value less than 0.05 or 0.005 was denoted by a single or double asterisks (*), respectively. Identical treatment (only with MG132) and analysis were done for cells transiently expressing GFP-endobrevin or GFP alone (G) (UT, untreated; TR, MG132 treated). However, no significant difference was observed. Malin and laforin co-localize in cytoplasm (H): COS-7 cells transiently overexpressing GFP-tagged malin and Myc-tagged laforin were visualized for their co-localization in the absence (untreated) or presence of MG132 (H) as indicated. Anti-myc antibody was used to detect laforin protein. Cells were counterstained with DAPI (blue color) to reveal the nucleus (merge panel). Aggregation of endogenous malin and laforin upon MG132 treatment (I and J). HEK293 cells treated with 5 µM of MG132 for 24 h and were stained using anti-malin and anti-{gamma}-tubulin antibodies (I) or anti-laforin and anti-{gamma}-tubulin antibodies (J). Arrows indicate centrosomal staining by the anti-{gamma}-tubulin antibody. For both combinations, the cells were counterstained with DAPI (blue color; merge panel). Scale bar, 10 µm (assembled images in each group are of the same magnification).

 
In addition to the diffuse staining pattern, we found that malin and laforin showed several small spherical inclusions throughout the cytoplasm in some cells. The relative frequency of inclusion formation was <8% of transiently transfected cells. We therefore examined whether the subcellular localization pattern of malin or laforin is altered when cells are subjected to various stress-inducing agents under transient transfection conditions (Fig. 1E and F). These include MG132, lactacystin (reversible and non-reversible proteasomal inhibitors, respectively), sorbitol (osmotic stress), tunicamycin (unfolded protein response), methyl methanesulfonate (MMS; DNA damaging agent) and hydrogen peroxide (oxidative stress). For this, COS-7 cells were exposed to each one of these agents for 16 h, 12 h post-transfection. We found that the expression pattern of laforin and malin was significantly altered when the cells were treated with the proteasomal inhibitors, MG132 or lactacystin (Fig. 1A, E and F). On both conditions, a majority of cells showed perinuclear cytoplasmic inclusions for laforin or malin protein, suggesting that formation of these inclusions is due to the proteasomal dysfunction rather than the unique effect of an individual drug (Fig. 1A, E and F). Strikingly, malin formed larger inclusions, whereas laforin showed relatively smaller and diffused inclusion primarily in the perinuclear space. Cells having inclusions showed more intense staining when compared with untreated cells, suggesting that there was an increase in the cellular levels of malin and laforin as a result of proteasome impairment. This suggestion was further strengthened by our western analysis that the level of malin and laforin was indeed increased in cells treated with MG132 (Fig. 1D). Similar to COS-7 cells, malin or laforin inclusions were also found in SH-SY5Y or HEK293 cells when treated with MG132 (Fig. 1B and Supplementary Material, Fig. S1A), implying that inclusion formation was not restricted to a specific cell line but was due to a specific response to proteasome impairment. No significant difference in localization pattern was observed for laforin or malin when cells were exposed to sorbitol, tunicamycin, MMS or hydrogen peroxide (Fig. 1E and F). The inclusion-formation property of laforin or malin was not affected by the type of the fusion tags used (GFP, FLAG or Myc or no tag) or their location in the fusion protein (N- or C-terminal) (Supplementary Material, Fig. S1B and D).

Proteasomal inhibitors induce selective aggregation of laforin and malin
The perinuclear inclusions seen for laforin and malin under proteasomal inhibition are reminiscent of aggresomes demonstrated for the cystic fibrosis transmembrane conductance regulator (CFTR) protein (24). In order to check whether or not the inclusions formed by overexpression of malin or laforin represent a selective process of protein aggregation as a result of proteasomal blockade, we examined the subcellular localization of GFP–CFTR, GFP-endobrevin or GFP alone by overexpressing them individually in COS-7 cells either in the presence or absence of MG132 (Fig. 1C). Although the characteristic perinuclear aggregates are seen for the CFTR under proteasomal inhibition, no obvious difference in the localization pattern was seen for endobrevin (an early endosomal membrane protein) (25) (Fig. 1C and G) or the GFP (Fig. 1G). Taken together, our results suggest that the formation of inclusion by laforin and malin due to the proteasomal inhibition is a selective process and might use a similar mechanism proposed for the CTFR-mediated aggresomes.

Laforin–malin co-expression does not alter their subcellular localization
We have demonstrated earlier that laforin is localized in the rough endoplasmic reticulum (8). Although laforin was shown to be a substrate for malin (10,11), whether or not the localization pattern of malin or laforin is altered when expressed together was not addressed. We therefore co-expressed laforin and malin using expression constructs in cell lines (Fig. 1H). Consistent with our previous report (8), laforin showed reticular cytoplasmic localization in a great majority of cells, and this pattern does not seem to be affected by the presence of malin or the cell line used (Fig. 1H and Supplementary Material, Fig. S1A). However, we find the expression level of laforin to be lower when co-expressed with malin (Supplementary Material, Fig. S1F), an observation that strengthens recent findings which demonstrated that laforin is ubiquitinated and degraded by malin (10,11). Similarly, we did not find any appreciable difference in the cellular localization of malin when co-expressed with laforin (Fig. 1H). We next examined whether the MG132-induced aggregates of malin and laforin co-localize when expressed together. For this, cells co-expressing malin and laforin were treated with MG132 and processed for immunostaining. As shown in Fig. 1H, laforin, which normally form smaller inclusions when compared with malin, was recruited into the larger aggregates that appear to represent homogenous structures.

Proteasomal inhibitor induces aggregation of endogenous laforin and malin
In order to further confirm that the formation of aggregates by laforin and malin is a selective process and is not due to their overexpression in transient conditions, we checked whether endogenous laforin and malin form aggregates in response to MG132 treatment. We have chosen HEK293 cell line for this purpose and confirmed, by RT–PCR, that both EPM2A and NHLRC1 genes are expressed (data not shown). By indirect immunofluorescence staining, we show that endogenous malin and laforin form aggresomes upon MG132 treatment (Fig. 1I). The specificity of the anti-malin antibody was confirmed by double staining cells that overexpressed malin, by western analysis and by pre-blocking antibodies with the antigen (Supplementary Material, Fig. S1C and F).

Laforin and malin inclusions exhibit aggresome-like properties
Since laforin and malin inclusions showed perinuclear distribution and are induced only by proteasomal inhibition, we then examined whether they display aggresome-like microtubule-associated properties, established for the CFTR protein (24). COS-7 cells were transiently transfected with laforin or malin expression constructs and treated with MG132, nocodozole (a microtubule disruption agent), both or none before fixation and processed for immunofluroscence staining (Fig. 2 and Supplementary Material, Fig. S2). The cells were co-stained for the {alpha}-tubulin protein to confirm the disruption of microtubules (Fig. 2A–D and Supplementary Material, Fig. S2A–D). Normal pattern of {alpha}-tubulin was observed when cells were co-expressed with laforin or malin. Malin and laforin showed normal distribution even when the cells were treated with nocodazole; however, microtubule organization was found to be defective as expected (Fig. 2C and Supplementary Material, Fig. S2C). In contrast, {alpha}-tubulin appeared to have been recruited into the laforin/malin inclusions upon MG132 treatment (Fig. 2B and Supplementary Material, Fig. S2B). Such {alpha}-tubulin-positive inclusions were not seen in cells that did not express laforin or malin. However, treatment of cells with both the drugs markedly abrogated the MG132-induced accumulation of malin or laforin into large perinuclear aggresomes, although relatively smaller aggregates were seen all over the cytoplasm (Fig. 2D and Supplementary Material, Fig. S2D). Taken together, these data suggest that microtubule networks are required for the formation of laforin- or malin-containing aggresomes.


Figure 2
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  		Figure 2. Intact microtubule is required for the formation of perinuclear aggregates of malin. COS-7 cells transiently expressing GFP-tagged malin were grown in the absence (A) or presence of 20 µM MG132 (B) or 10 µg/ml nocodazole (noco) (C) or both (D) for 12 h. Cells were fixed in methanol and immunostained with an antibody against {alpha}-tubulin (A–D; red color). Similarly, cells transiently expressing GFP-malin were co-stained for {gamma}-tubulin without (E) or after treatment with MG132 (F). Overlays of each set include DAPI staining (blue color). Arrows indicate the centrosomal staining by the anti-{gamma}-tubulin antibody. Scale bar, 10 µm (assembled images in each group are of the same magnification).

 
One of the characteristic features of the aggresome includes its formation at the microtubule organizing center (MTOC) or the centrosome (24). To examine whether the peri-nuclear aggregates of laforin and malin proteins are similar in structure to aggresomes, we co-stained these aggregates with {gamma}-tubulin, a marker for the MTOC. Immunofluorescence imaging revealed that the signals of {gamma}-tubulin and aggregates of malin/laforin are co-localized, thereby indicating a close physical relationship between the aggresome and the MTOC (Fig. 2E and F and Supplementary Material, Fig. S2E and F). The presence of aggresome at the MTOC suggests a direct involvement of microtubules in the formation of the centrosomal structure. Aggresome formed by endogenous laforin or malin also showed positive staining for {gamma}-tubulin (Fig. 1I–J).

Critical players of ubiquitin-proteasome pathway co-localize with aggresomes of laforin and malin
Malin is an E3 ubiquitin ligase, which ubiquitinates and promotes the degradation of laforin (10,11). We therefore checked whether some of the critical players of the ubiquitin-proteasome pathway are localized within aggresome in cells over-expressing laforin or malin. In the absence of MG132, the anti-ubiquitin antibody showed diffused staining in the nucleus and cytoplasm, with very little overlap with the laforin or malin signals (Supplementary Material, Fig. S3A and B). After the addition of MG132, however, there was a distinctive change in the localization of ubiquitin, which was predominantly co-localizing with the aggresome of malin or laforin (Fig. 3A and B). We have also analyzed the localization of GRP78/Bip (an ER resident chaperone), and UbcH5a (an E2-conjugating enzyme known to interact with malin (10)) within laforin- or malin-containing aggresomes. Compared with its staining pattern in the absence of MG132, UbcH5a and GRP78 redistributed to aggresomes in proteasome inhibitor-treated cells and co-localized with malin- or laforin aggregates (Fig. 3E–H and Supplementary Material, Fig. S3E–H). Similarly, although proteasome localization was observed within the aggresome containing laforin or malin, the proteasomal staining pattern in cells that were not treated with MG132 was more diffused and cytoplasmic (Fig. 3C and D and Supplementary Material, Fig. S3C and D). We also assessed the distribution of mitochondria in the cells harboring aggresome because disruption of mitochondria is known to be associated with protein aggregates (26–28). For both malin and laforin, immunofluorescence staining revealed mitochondrial clustering around the aggresome in the MG132-treated cells, whereas in the untreated cells, they were distinct from the distribution of laforin or malin (Fig. 3I and J and Supplementary Material, Fig. S3I and J).


Figure 3
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  		Figure 3. Laforin and malin inclusions exhibit aggresome-like properties: COS-7 cells transiently expressing GFP-tagged malin (A, C, E, G and I) or laforin (B, D, F, H and J) were cultured in the presence of 5 µm of MG132 for 24 h and immunostained individually with antibodies for following proteins: ubiquitin (A and B), 20S subunit of proteasome (C and D), ubiquitin-conjugating enzyme UbcH5a (E and F), ER chaperone Bip/GRP78 (G and H) and a 60 kDa mitochondrial resident protein (I and J). Overlays of each set include DAPI staining (merge panel; blue color). Scale bar, 10 µm (assembled images in each group are of the same magnification).

 
Effect of LD-associated mutations on aggresome formation
Since the formation of aggresome appears to be the intrinsic property of LD proteins, we checked whether LD-associated mutants respond differently when compared with wild-type protein in our assay conditions. We selected four missense mutants and two deletion mutants for malin for the expression study (Fig. 4A). Association of these mutations with LD phenotype was confirmed in a recent report from our group (19). The effect of LD-associated malin mutations on cellular localization was more diverse. Mutations W219R, delF216_D233 and V359fs30, which affect the NHL repeats, had significantly increased number of cells forming the inclusion when compared with wild-type malin (Fig. 4B). For mutants E91K and P218S, the number of cells having aggregates was comparable to that of wild-type or the C26S mutant (Fig. 4B). Western blotting of cell lysates did not show any significant difference in the level of expression between mutants that form inclusions when compared with those that do not (Supplementary Material, Fig. S4B). Thus, the formation of inclusions does not seem to be associated with differential expression level for malin mutants. The size of the inclusions formed by the mutant forms of malin was relatively smaller when compared with the MG132-induced aggresome of the wild-type malin. In the presence of MG132, however, all mutant forms resulted in the production of larger inclusions in a great majority of cells (>75%), similar to that of wild-type malin (Supplementary Material, Fig. S4G).


Figure 4
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  		Figure 4. Effect of LD-associated mutations on subcellular localization of malin or laforin. Schematic illustration on domain organization of malin (A) and laforin (C) proteins and positions of mutations (arrow heads) that are tested in this study are shown (dotted lines for the malin mutant delF216_D233 denote the deleted region) (WT, wild-type) (CBD, carbohydrate-binding domain; DSPD, dual-specificity phosphatase domain; RING, RING finger domain; NHL, NHL-repeat domains). Bar diagrams show percent cells, expressing each of the six mutants tested for malin (B) and four mutants for laforin (D), developing aggresome in the absence of MG132. The values represent the mean average of three independent transfections, with a minimum of 300 transfected cells scored each time. Error bars indicate the standard error of the mean. A difference in the number of cells having laforin or malin aggregates between the mutants (as indicated) and the wild-type (WT) group was calculated for statistical significance by paired t-test. A P-value less than 0.05 or 0.005 was denoted by a single or double asterisks (*), respectively. Representative figures for the subcellular distribution of malin mutants C26S and W219R (E) and laforin mutants E28L and Q293L (F) under transient expression conditions in COS-7 cells. Aggregates formed by malin (G) or laforin (H) mutants are positive for ubiquitin. Representative mutants (as indicated) are transiently expressed in COS-7 cells in the absence of MG132 and double stained with anti-ubiquitin antibody. Overlapping signals are identified by arrows (see also Supplementary Material, Fig. S4C–D). Scale bar, 10 µm (assembled images in each group are of the same magnification).

 
We have shown earlier that some of the laforin mutants form large cytoplasmic inclusions when transiently expressed (8,29). Here we re-analyzed four laforin mutants to quantify the number of cells having inclusions and to see whether they display aggresome-like properties. A majority of the cells (>85%) expressing laforin mutants Q293L and G279S formed large perinuclear aggregates when compared with the wild-type laforin (Fig. 4D). On the other hand, mutants E28L and R108C, located in the carbohydrate-binding domain of laforin, did not show appreciable difference in the number of cells having inclusion (Fig. 4D). Strikingly, inclusions resulting from the mutants are large in size when compared with the MG132-induced aggresome for laforin (Fig. 4F). All four mutants formed larger inclusions in a great majority of cells upon MG132 treatment (Supplementary Material, Fig. S4H).

The inclusions of mutant forms of malin or laforin were positive for ubiquitin and {gamma}-tubulin, suggesting that they show properties of aggresome even in the absence of proteasome inhibitor (Fig. 4G and H; Supplementary Material, Fig. S4C–F).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
We demonstrate here that proteasomal inhibition induces centrosomal accumulation of laforin and malin, the two proteins that are defective in LD. Such cytoplasmic accumulations, known as aggresomes, are observed for many proteins involved in neurodegenerative diseases and are formed from aggregated proteins by retrograde transport on microtubules to the MTOC (30,31). The recruitment of laforin and/or malin into aggresome appears to be a specific response of the cell to inhibition of proteasomal function, as none of the other stress-inducing agents used in the present study had any significant effect on the sub-cellular localization of laforin or malin. The centrosomal accumulation was observed for the LD proteins when overexpressed separately, or together, in more than one cell lines. Aggresomes were also observed for endogenous malin and laforin in HEK293 cells, suggesting that the MG132-induced aggregates of malin/laforin seen in transient transfection conditions are not because of their overexpression. Overexpression of these proteins, however, does result in a small fraction of transfected cells developing smaller aggregates even in the absence of proteasome inhibitor, suggesting that inclusion formation may occur naturally in cells expressing a very high level of laforin or malin. As originally suggested for the CFTR protein (24), the formation of inclusion appears to be an inherent property of laforin and malin, as overexpression of GFP or the endobrevin proteins did not produce any inclusions even after the inhibition of proteasome activity. These results clearly show that not all proteins go to centrosomal region when the proteasome functions are inhibited.

The MTOC contains a variety of proteins, such as proteins responsible for the proteasome-proteolytic pathway, cell-cycle regulatory system and for the nucleation of microtubules (24,30,32–34). The {gamma}-tubulin protein is one of the components of the MTOC and is required for the structure and normal function of the centrosome (32,35). Recent studies have shown that centrosomes are also required for the removal of abnormal proteins, as accumulation of misfolded proteins leads to a significant increase of {gamma}-tubulin in the centrosome (24,31,33). This recruitment process however requires intact microtubule network (24,36). We show here that the formation of aggresomes for malin and laforin proteins resulted in the redistribution of {gamma}-tubulin and disrupted the normal distribution of the cytoskeletal protein, {alpha}-tubulin. When microtubules were depolymerized by the addition of nocodazole, the formation of larger aggresome was abrogated. Aggresome formation is known to be blocked by drugs that depolymerize microtubules and by the expression of the microtubule-binding protein dynamitin (24,36,37). Thus, a critical role for dynein-based retrograde transport on microtubules has been proposed for the formation of aggresome in mammalian cells (37,38). In light of these finding, our observations on the effect of nocodozole on MG132-induced aggresomes strongly support the conclusion that aggresomes are formed by the retrograde transport of malin/laforin on microtubules and that the formation of aggresome is dependent on the microtubule function.

We show here that the MG132-induced aggresome of malin/laforin stained positive for proteasome, ubiquitin and the ER chaperone Bip/GRP78. These observations are consistent with the emerging notion that aggreasomes recruit a number of components, such as chaperones, ubiquitination enzymes and proteasome machinery, to help in the clearance of aggregated proteins (16,31). Indeed, overexpression of a cytosolic chaperone was shown to prevent aggresome formation, suggesting that the clearance of aggregated protein is limited by insufficient amount of chaperone to unfold substrates for proteasomal degradation (39,40). Substrates thus unfolded are likely to be ubiquitinated by E3 ligases, as proteasomal substrates are often ubiquitinated (13,15). Since E3 ubiquitin ligases display substrate specificity, it is likely that the recruitment of E3 enzymes to aggresome perhaps reflects a cellular response to a distinct set of proteins and is not a consequence of aggresome formation (13,14,16,17,31). It could therefore be suggested that malin is recruited to aggresome to provide a subcellular locale for this E3 enzyme to ubiquitinate and degrade its substrates whose cellular levels have increased because of the MG132 treatment. This event is likely to be facilitated by ubiquitin-conjugating enzymes because UbcH5a, which aids malin in the ubiquitination process (10), was also recruited to the aggresome. Thus, the centrosomal aggregation of malin may enhance ubiquitination of its substrates; therefore, they can be efficiently degraded. The clustering of mitochondria around the aggresome may be to provide the required ATP to the UPS, because impaired mitochondrial function is known to induce aggresomes in otherwise normal condition (26–28).

Although the proposed role for malin is consistent with the ubiquitin-proteasome functions, what could be the possible reason for the recruitment of laforin into aggresome upon MG132 treatment? One obvious explanation is that laforin is a substrate for malin and therefore recruited to aggresome for its clearance by malin. It is also possible that delivering laforin protein to the MTOC might promote sequestration of aggregates/unfolded proteins into aggresomes by localized regulation of critical cellular signaling mechanisms. For example, it is known that the activation of stress kinases strongly promotes the formation of aggresome for misfolded proteins (41,42). Thus, laforin is likely to be a novel component of signaling circuits and may perhaps overlap with the pathway regulated by malin. It may be noted that the MG132-induced aggresomes of laforin are smaller in size and are dispersed in cytoplasm when compared with larger aggresome of malin. When co-expressed, however, both malin and laforin are recruited into large peri-nuclear aggresome structures. Thus, it is likely that co-expression of laforin and malin leads to aggregation of both proteins into single aggregates and that this may be required for the function of the aggresome. Clearly, the cellular functions of laforin in the formation and functions of aggresome need to be studied further.

To date, more than 40 distinct mutations have been described for each of the two genes defective in LD; EPM2A and NHLRC1 (2,43). Observed mutations include missense, nonsense and frameshift point mutations as well as exon deletions (2). Despite such remarkable allelic heterogeneity, no significant difference in the clinical manifestations among LD patients carrying different forms of mutations in the EPM2A or NHLRC1 gene has been observed. This could perhaps mean that amino acid substitutions, resulting from missense mutation, are as deleterious to the protein function as are the exonic deletions or the truncation due to frameshift/nonsense mutations. Our work demonstrates that malin and laforin mutants display variable degrees of aggregation when individually expressed even in the absence of proteasome inhibitor. For example, malin mutant delF216_D233 (amino acid deletion), W219R (missense mutation) or V359fs30 (frameshift mutation) were more prone to aggregate than the three other mutants tested. Significantly, mutations that are located outside the RING domain had higher propensity to induce protein aggregates. For laforin, on the contrary, mutations affecting the phosphatase domain are more likely to form aggregates (present study, 8, 29). It has been shown for Parkin, an E3 ubiquitin ligase defective in Parkinson's disease, that a majority of the mutants tested for their subcellular localization formed aggregates and nearly half in this group had mutations within the RING finger domains (44). Therefore, it would be of importance to test whether or not other RING mutants reported in the literature affect the subcellular localization of malin. Nevertheless, the aggregation properties of laforin or malin mutants are similar to those reported for Parkin (21,44); they were positive for ubiquitin, proteasome and {gamma}-tubulin, suggesting that the mutants were misfolded proteins set for degradation. Taken together, our results suggest that alterations in the subcellular localization of LD proteins may underlie the molecular basis of loss of malin or laforin function for these mutations. Clearly, structural studies would provide insights into the effect of mutations on conformational changes of the resulting protein.

The presence of proteinaceous aggregates, similar to the aggresome described here, in a number of neurodegenerative disorders suggests that aggresomes are linked to pathogenesis (13–16). It is highly probable that pathological conditions that affect the UPS might lead to the formation of insoluble and/or toxic forms of protein aggregates when there is a change in the equilibrium between the production and degradation of proteins (15,16). Our results demonstrate that centrosomal accumulation of malin, possibly with the help of laforin, may enhance the ubiquitination of its substrates and facilitate their efficient degradation by proteasome. Defects in malin (or laforin) may thus lead to increased levels of unwanted/undesired proteins in the neuron. Although proteinaceous inclusions are not observed in LD, the Lafora polyglucosan bodies are known to contain (up to 10%) non-degradable proteins (5,6). Lafora bodies are seen when any one of the two LD proteins is defective (11,19,29). Although Lafora inclusions resulting from laforin deficiency are ubiquitin-positive, those that develop because of malin deficiency are devoid of ubiquitin (5,11). This observation suggests that proteins that are trapped in the Lafora bodies are substrates for malin and therefore not ubiquitinated when malin is defective. Therefore, it is likely that laforin and malin together regulate the cellular levels of critical players of glycogen metabolism, and the loss of either laforin or malin leads to impaired degradation of their substrates and their abnormal functions. Curiously, laforin is known to complex with critical players of glycogen metabolism (11,45,46). Laforin and its substrate glycogen synthase kinase 3ß are found to be trapped in the Lafora inclusions (11,46,47). Remarkably, a protein similar to laforin in plants was recently shown to initiate critical steps in the starch degradation, and loss of this proteins results in excessive starch accumulation (48,49). Because Lafora bodies are starch-like polyglucosans (50) and because players involved in glycogen synthesis appear to be unaltered in LD (51), it would be of interest therefore to explore the cooperative regulation of critical players involved in the glycogen degradative pathway by laforin and malin. In summary, our results suggest that both malin and laforin are involved in the ubiquitin-proteasomal pathway, and the primary trigger in LD is likely to be the defects in protein degradation. This notion should now be experimentally tested in animal models.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Expression constructs
Malin coding sequence was amplified by polymerase chain reaction from genomic DNA by using Pfu DNA polymerase (Fermentas), purified by agarose gel electrophoresis and ligated in-frame with the GFP coding sequence into the pEGFPC2 vector. This clone would encode a fusion malin with the GFP at its N-terminus (GFP-malin). The malin coding sequence was cloned into the pcDNA3 vector either in-frame or out of frame for an Myc-epitope coding region to create constructs coding for malin with C-terminal Myc tag (Myc-malin) and without any tag, respectively. For malin mutants, the coding regions were amplified from the genomic DNA of LD patients for whom these mutations have been established (19). The coding sequence of endobrevin was amplified from a human cDNA and cloned into the pEGFPC2 vector. All clones were sequence-confirmed. Primer sequences used for the amplification of malin or endobrevin coding sequence are available on request. Laforin expression constructs are described in our previous publications (8). The GFP–CFTR construct was kindly provided by Prof. Ron Kopito.

Antibodies
The following antibodies were used in immunofluorescence studies. Mouse monoclonal antibodies for GFP (clone GFP 20), {alpha}-tubulin (clone DM 1A), {gamma}-tubulin (clone GTU-88) and rabbit polyclonal antibodies for actin and FLAG epitope were obtained from Sigma-Aldrich Chemicals Pvt Ltd (India). Rabbit polyclonal antibodies for Myc-epitope was obtained from Cell Signaling Technology (USA). Rabbit polyclonal antibodies for the 20S proteasome and ubiquitin were purchased from Calbiochem (USA). Goat polyclonal antibody for BIP/GRP78 was purchased from Santa Cruz Biotechnology (USA). Mouse anti-human mitochondria antibody was purchased from Chemicon International (USA). Goat polyclonal antibody for malin was obtained from Everest Biotech (UK) and rabbit polyclonal antibody for laforin was described in our previous study (8). Secondary antibodies were obtained from Jackson Immuno Research (USA).

Cell culture and transfection
COS-7, SH-SY5Y and HEK293 cell lines were maintained in Dulbecco's modified Eagle's medium (Sigma-Aldrich). All cells were grown at 37°C in 5% CO2 and supplemented with 10% (vol/vol) fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin. Transfections were performed using LipofectAMINE 2000 transfection reagent (Invitrogen, USA) according to the manufacturer's protocol. Cells were cultured for 24 h after transfection.

Treatment
Unless stated otherwise, stress-inducing reagents were added to cell cultures 16 h post-transfection and cultured for 12 h. Such reagents included 20 µM MG132 (Calbiochem), 10 µg/ml lactacystin, 400 µM hydrogen peroxide, 300 mM sorbitol, 10 µg/ml tunicamycin, 100 µg/ml MMS, 400 µM hydrogen peroxide or 10 µg/ml nocodazole (all obtained from Sigma-Aldrich). For aggregate formation and double staining, 5 µM MG132 was added to the medium 6 h post-transfection and cultured for 24 h. Control experiments using the resuspension solvents dimethyl sulfoxide (DMSO) were performed where appropriate.

Immunocytochemistry
Cells, grown on gelatin-coated sterile glass coverslips, were fixed and processed for immunofluorescence microscopy essentially as described by Ganesh et al. (8). Cells were fixed in methanol for cytosketal staining or in 2% paraformaldehyde for other components/proteins. For nuclear staining, fixed cells were incubated with 10 µM 4',6-diamidino-2-phenylindole (DAPI) for 5 min subsequent to secondary antibody incubation. Fluorescence images were obtained using a fluorescence microscope (AxioScope 2 plus, Carl Zeiss, Germany) with 40x objective lens and were processed using Axiovision software (Carl Zeiss, Germany). The number of transfected cells with or without inclusion bodies was counted for a minimum of 300 transfected cells. Experiments were repeated at least thrice, and counts were made in a blinded manner.

Immunoblotting
Protein samples were run on a 10% SDS–PAGE and transferred onto a nitrocellulose filter (MDI, India) as described previously (8). After blocking with 5% non-fat dry milk powder, the membranes were processed through sequential incubations with primary antibody followed by secondary antibody. Immunoreactive proteins on the filter were visualized using a chemiluminscent detection kit (SuperSignal West PICO, Pierce, USA).


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


   ACKNOWLEDGEMENTS
 
The authors wish to thank Dr Ron Kopito for the gift of the CFTR expression constructs. This work was supported in part by a research grant from the Board of Radiation and Nuclear Sciences, Department of Atomic Energy, Government of India (S.G.) and a collaborative research grant from the RIKEN Brain Science Institute, Japan (S.G. and K.Y.). S.M. and D.D. were supported by research fellowships from the Council of Scientific and Industrial Research, Government of India.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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