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Human Molecular Genetics Advance Access originally published online on October 21, 2003
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Human Molecular Genetics, 2003, Vol. 12, No. 24 3231-3244
DOI: 10.1093/hmg/ddg346
© 2003 Oxford University Press

Autophagy regulates the processing of amino terminal huntingtin fragments

Zheng-Hong Qin1, Yumei Wang1, Kimberly B. Kegel1, Aleksey Kazantsev1, Barbara L. Apostol2, Leslie Michels Thompson2, Jennifer Yoder1, Neil Aronin3 and Marian DiFiglia1,*

1Laboratory of Cellular Neurobiology, Massachusetts General Hospital and Harvard Medical School, 16th Street, Bldg 114, Rm 2125, Charlestown, MA 02129, USA, 2Department of Psychiatry and Human Behavior, University of California, Irvine, CA 92697, USA and 3Department of Medicine and Cell Biology, University of Massachusetts Medical Center, Worcester, MA 01655, USA

Received May 13, 2003; Revised August 12, 2003; Accepted October 9, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The N-terminus of mutant huntingtin (htt) has a polyglutamine expansion and forms neuronal aggregates in the brain of Huntington's disease (HD) patients. Htt expression in vitro activates autophagy, but it is unclear whether autophagic/lysosomal pathways process htt, especially N-terminal htt fragments. We explored the role of autophagy in htt processing in three cell lines, clonal striatal cells, PC12 cells and rodent embryonic cells lacking cathepsin D. Blocking autophagy raised levels of exogenously expressed htt1–287 or 1–969, reduced cell viability and increased the number of cells bearing mutant htt aggregates. Stimulating autophagy promoted htt degradation, including breakdown of caspase cleaved N-terminal htt fragments. Htt expression increased levels of the lysosomal enzyme cathepsin D by an autophagy-dependent pathway. Cells without cathepsin D accumulated more N-terminal htt fragments and cells with cathepsin D were more efficient in degrading wt htt than mutant htt in vitro. These results suggest that autophagy plays a critical role in the degradation of N-terminal htt. Altered processing of mutant htt by autophagy and cathepsin D may contribute to HD pathogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Two main proteolytic pathways are used by cells to degrade cellular proteins. One pathway involves ubiquitin and the proteasome; proteins, including those misfolded, are tagged by ubiquitin and then degraded by the proteasome (1,2). The other pathway is autophagy (3,4). Autophagy is a regulated process that is activated for bulk removal of cellular proteins and organelles (5,6). Proteins are targeted and transported to membrane-enclosed vesicles. Vesicles are fused with lysosomes and their contents degraded by lysosomal enzymes, cathepsin D prominent among them (6,7). Autophagy and proteasomes may work together to degrade misfolded proteins that have been retained in the endoplasmic reticulum (8). Autophagy in neurodegenerative diseases, including Parkinson's, Alzheimer's and prion diseases (9–12), can trigger a form of cell death distinct from apoptosis (13). We showed that expression of wild type or mutant huntingtin (htt) in clonal striatal cells (x57 cells) induced autophagy and caused htt to associate with autophagosomes, which fused into larger structures (htt bodies) (14). In striatal cells derived from Huntington's disease (HD) transgenic mice expressing mutant huntingtin exon 1, oxidative stress induced by dopamine stimulated autophagy (15). Inhibitors and stimulators of autophagy can regulate levels of N-terminal mutant htt (N-htt) in cells and cell viability (16,17). These studies suggest that autophagy may be involved in normal processing of htt in cells and that autophagy induced by the presence of mutant htt could be activated in the HD brain.

Huntingtin is a large protein consisting of 3144 amino acids. A pathological hallmark of HD is the appearance of intranuclear and cytoplasmic inclusions containing fragments of N-htt in affected regions of the brain (18). Mutant N-htt produces nuclear and cytoplasmic inclusions in the mouse brain and disrupts cell function in vitro (19–21). Therefore, the production and accumulation of N-htt fragments may be a critical part of the pathogenic process. Caspase-3 and calpain are proteases that cleave htt to produce stable N-htt fragments (22–24). Increases in cathepsin D (cat D) and H activity have been found in affected areas in HD brain (25). Because autophagy might contribute to htt cleavage, we examined the role of autophagy and the activity of the autophagic/lysosomal enzyme cathepsin D in the degradation of htt. The results suggest that autophagy and cathepsin D are involved in the catabolism of N-htt fragments and that polyglutamine expansion impairs htt degradation by cathepsin D.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Autophagy regulates levels of intact htt1-287 andhtt1-969 and caspase-cleaved N-htt fragments in clonal striatal cells (x57)
3-methyladenine (3-MA) selectively inhibits class III PI3 kinase (26,27), which is the mammalian homolog of yeast VPS34, a vacuole sorting protein essential for autophagy and for protein sorting from the Golgi to the vacuole/lysosome (28). Human VPS34 may be required for the internalization of vesicles within multivesicular bodies (29). 3-MA inhibits autophagy in many cell lines (16,27,30,31). We studied the effects of 3-MA in x57 cells expressing a Flag-tagged truncated htt encoding htt1–287 amino acids with 18 glutamines (Q) (wt) or 100 Q (mutant). Six hours after transfection of FH287-18 or FH287-100 cDNA, addition of 3-MA (10 mM) to culture medium for 8 h increased the levels of wild-type htt1–287 amino acids protein (141±6.7% of control, M±SE, P<0.05, one group t-test) and mutant htt1–287 (158±25.4% of control, M±SE; n=3) (Fig. 1A, left). 3-MA treatment for 18 h significantly reduced the density of FLAG-labeled cells to 54% of control for wild-type htt (P<0.03, Student's t-test, n=3) and to 62% of control for mutant htt (P<0.05, Student's t-test, n=3) (Fig. 1C). The drug showed no apparent effect on cell survival by MTT assay (see below and Fig. 3C) as Flag-positive cells account for a small percent of total cells. In cells expressing mutant htt1–287, 3-MA treatment increased the proportion of cells with Flag-labeled aggregates from 7.8 to 17.1% (M±SE, P<0.001, Student's t-test, n=6) (Fig. 1D). Thus, in the Flag-htt expressing clonal striatal cells, inhibition of autophagy with 3-MA raised levels of exogenous htt and mutant htt aggregates and reduced viability. To confirm the effects of inhibiting autophagy on processing of exogenously expressed htt, we treated cells with bafilomycin A1 (BAF), which is a specific inhibitor of the vacuolar proton pump that blocks maturation of autophagic vacuoles and acidification of lysosomes (32). Similar to the effects with 3-MA, BAF treatment for 8 h, starting 6 h after transfection, raised levels of intact htt1–287-100 (129% of control; n=2) (Fig. 1B). The findings with 3-MA and BAF suggest that autophagy promotes the degradation of wild type and mutant htt1–287.



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  		Figure. 1 Effects of 3-MA and bafilomycin (BAF) on htt expression and the formation of mutant htt aggregates in clonal striatal cells (x57 cells). x57 cells were transfected with FH287-18 or FH287-100. Six hours after transfection, cells were incubated with medium with or without 3-MA (10 mM) and harvested 8 h later for analysis. (A) Western blots show exogenous wt htt (left arrow), detected with anti-htt antibody Ab1 and mutant htt (right arrowhead) detected using monoclonal antibody 1C2. Ab1 detects the first 1–17 amino acids in htt. 1C2 preferentially recognizes expanded polyglutamine peptides. The intact wt htt (arrow, top left) and mutant htt (arrowhead) are identified. The less abundant wt htt fragment is a degradation product of wild-type htt1–287. The larger less abundant mutant htt product may be a modified form of mutant htt1–287. No breakdown products were detected for mutant htt in this assay. Results from three experiments were quantified using SigmaPlot Pro 4 (see Results). 3-MA treatment increases levels of intact wt (arrow top left) and mutant htt (arrowhead). Actin levels are unchanged. The results in (A) are representative of three experiments. (B) x57 cells were transfected with FH287-100. Cells were incubated with medium with or without BAF (200 nM) 6 h after transfection and processed for SDS–PAGE and western blot analysis using 1C2 monoclonal antibody. BAF treatment increases levels of intact mutant htt (arrowhead). Actin levels are unchanged. The results in (B) are representative of two experiments. (C) x57 cells were transfected with FH287-18 or FH287-100. Cells were incubated with medium with or without 3-MA (10 mM) 6 h after transfection and fixed and processed for Flag immunofluorescence 24 h after htt transfection. Cells were visualized using epifluorescence microscopy. The number of Flag-labeled cells is significantly reduced by 3-MA treatment (height of bars=Mean±SE, Student's t-test, *P<0.03 for FH287-18, n=3 and *P<0.005 for FH287-100, n=3). (D) x57 cells were transfected with FH287-100 with or without 3-MA using the same paradigm as in (C). Htt aggregates were visualized with anti-Flag antibody M5 and examined with a confocal microscope. One thousand Flag-htt positive cells were counted and six slides were analyzed in each group. 3-MA treatment significantly increases the number of cells bearing aggregates (bar=mean±SE). Statistical analysis was carried out using the Student's t-test. ***P<0.001.

 


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  		Figure 3. Effects of 3-MA in clonal striatal cells (x57) expressing htt. (A) Western blot analysis with Ab 1 antibody of lysates from x57 cells. The cells were transfected for 24 h with FH969-18 or mutants of FH969-18 that alter (D) to (A) at amino acids 513 or 552 (the caspase-3 cleavage sites). N-htt fragments were seen with molecular mass of about 70–90 kDa (lane 1 from left) and the levels were reduced by rapamycin (Rap, lane 2). The two smaller N-htt fragments (bracket) are absent when residue 513 is mutated. The smaller of the two bands may be a breakdown product. The mutation increases levels of two larger products (lane 3). The largest N-htt fragment (arrowhead) is not expressed when residue 552 is changed (lane 4). (B) Six hours after transfection, cells were incubated with medium with or without the autophagy inhibitor 3-MA (10 mM). Sixteen hours after treatment with 3-MA, cells were harvested to analyze htt levels or processed for immunofluorescence as described in Materials and Methods. On the left is a representative western blot of whole cell lysates, soluble fractions and detergent insoluble fractions from cells expressing FH969-18 or FH969-100. Htt was detected with Ab 1 antibody. Gray arrowheads indicate expressed htt1–969. Black arrowheads indicate N-htt fragments. The small arrow at the top identifies endogenous mouse htt. Results from three experiments were quantified using SigmaPlot Pro 4 (see Results). 3-MA treatment reduced levels of N-htt fragments in whole lysate and detergent insoluble fractions. Molecular mass standards are on the right. (C) Six hours after transfection of FH969-100, cells were incubated with medium with or without the autophagy inhibitor 3-MA (10 mM). Sixteen hours after treatment with 3-MA, cells were processed to analyze viability using MTT assay as described in Materials and Methods. There was no evidence for a significant effect of 3-MA on cell viability using the MTT assay. However, 3-MA treatment did reduce the number of Flag-labeled cells in the cultures (see Results). (D) Quantitative analysis shows that the number of cells with htt-labeled bodies (detected with anti-Flag antibody) is reduced by 3-MA treatment. One thousand Flag-htt positive cells were counted in each coverslip and six coverslips were analyzed in each group. Bar=mean±SE. Statistical analysis was carried out using the Student's t-test. ***P<0.001. (E) Cells were treated as described above and Flag-htt 1969 and N-htt fragments were immunoprecipitated with an anti-Flag M2-agarose as described in Materials and Methods. Omission of plasmid DNA during the transfection produced no exogenous Flag-htt (data not shown). More ubiquitin (bracketed area) is co-immunoprecipitated with Flag-htt after 3-MA treatment. The results were the same in two experiments.

 
To determine whether autophagy degraded larger htt products, 6 h after transfection with FH-969-18Q (wt) or FH-969-100Q (mutant), we exposed x57 cells to 3-MA for 8 h and then harvested the cells for preparation of detergent soluble and insoluble fractions or whole lysates 14 h post-transfection. 3-MA treatment increased wild-type (221.2±48% of control; M±SE, n=3) and mutant htt1–969 (314±64% of control; M±SE, n=3) in the detergent insoluble fraction (Fig. 2A). Whole cell lysates showed a small increase (about 10%) in the levels of wild-type htt1–969 and accumulation of cleaved N-terminal mutant htt fragments. The effects of BAF treatment on levels of mutant htt were assessed using the same paradigm. BAF raised levels of intact mutant htt (208±24% of control, M±SE, P<0.01, n=4, two group t-test) and cleaved N-terminal htt fragments (318±80% of control, M±SE, P<0.01, n=4, two group t-test) in the detergent insoluble fraction (Fig. 2B). The mutant htt fragments also increased after BAF treatment in the whole cell lysates (126% of control, n=2) and in the soluble fraction (144% of control, n=2). Cells harvested at 24 h post-transfection and exposed to 3-MA for only the last 8 h in culture produced substantial increases in the levels of wild-type (141.3±6.7% of control, M±SE, P<0.05, one group t-test) and mutant htt1–969 (157±16% of control, M±SE) in whole cell lysates (n=3). Similar increases in htt levels were also found in the detergent insoluble fraction (130.1±6% of control for wt htt1–969, M±SE, P<0.05; 142±11% of control for mutant htt1–969 P<0.06, M±SE, n=3) (Fig. 2C). In this paradigm, N-htt fragments generated from htt1–969 were recovered at 24 h post-transfection in the whole cell lysate and the detergent soluble fraction, but were not  changed by the 8 h treatment with 3-MA (Fig. 2C). Thus, both sizes of htt (htt1–287 and htt1–969) were degraded by autophagy. Observing a change in the levels of N-htt fragments generated from htt1–969 when autophagy is inhibited by 3-MA may depend upon the duration and time the inhibitor is introduced. Next we investigated the effects of autophagy on caspase dependent htt cleavage products.



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  		Figure 2. Effects of 3-MA and BAF on htt expression in clonal striatal cells (x57) expressing FH969-18 or FH969-100. Cells were exposed to fresh medium with or without 3-MA (10 mM) at 6 (A) or 16 h (C) after transfection or for BAF (200 nM) at 6 h (B) after transfection. After treatment, cells were harvested and htt expression was analyzed in cell lysates by western blot with Ab 1 antibody (A and C) or 1C2 (B). Densitometry was performed on the results (A, B and C) in three to four experiments using SigmaPlot Pro 4 (see Results). (A) 3-MA treatment from 6 to 14 h post-transfection increases htt1–969 (gray arrowheads) in detergent insoluble fractions and mutant htt fragments (black arrows) in the whole cell lysate. (B) BAF treatment from 6 to 14 h post-transfection increases intact mutant htt1–969 in detergent insoluble fraction and increases N-terminal htt fragments (indicated by bracket) in whole cell lysate, soluble fraction and detergent insoluble fraction. Actin levels are unchanged. (C) 3-MA treatment from 16 to 24 h post-transfection increases htt1–969 (gray arrowheads) in whole cell lysates and in detergent insoluble fractions. In this experimental paradigm the N-htt fragments (black arrowheads) are relatively unaffected by 3-MA treatment. Molecular mass standards are on the right in (A) and (B).

 
We had found that the pan-caspase inhibitor, Z-VAD-FMK, blocked generation of N-htt fragments from htt1–969 in x57 cells (33). These N-htt fragments did not form in MCF-7 cells, which are devoid of caspase-3 activity (23). These results suggested that N-htt fragments produced from htt1–969 might be products of caspase cleavage. Caspase-3 cleaves htt at amino acids 513 and 552 in vitro (22). To confirm that the N-htt fragments seen in x57 cells were cleaved by caspase-3, we generated mutations in FH969-18 to produce an amino acid substitution from D -> A at residue 513 or 552 (see methods). Expression of these mutant constructs in x57 cells abolished expression of N-htt fragments at the expected size (Fig. 3A, lanes 3 and 4), substantiating that the fragments generated in x57 cells from htt1–969 were products of caspase-3 cleavage at these sites.

We treated cells with rapamycin, which stimulates autophagy in vitro (34). Treatment with rapamycin (0.2 µg/ml) for 18 h, beginning 6 h after transfection of N-htt1–969 cDNA, significantly reduced levels of the caspase cleaved N-htt fragments, which were generated from wt htt1–969 (Fig. 3A, lane 1 versus 2, one group t-test, P<0.05 for upper band and P<0.01 for lower band). Rapamycin produced little change in intact htt1–969. Rapamycin had no effect on htt-induced activation of caspase-3 (data not shown). Thus, the reduction in the levels of caspase-3 cleaved htt fragments is unlikely to be caused by reduction in the availability of intact htt or inhibition of cleavage. These results suggest that autophagy can further degrade caspase-cleaved N-htt fragments.

We started 3-MA treatment before caspase cleavage had occurred and increased the duration of treatment to determine the effects of prolonged inhibition of autophagy on N-htt fragments generated from htt1–969. 3-MA was applied to x57 cells that were transfected with FH969-18 or FH969-100 cDNA for 24 h. The N-htt fragments accumulated to maximal levels 15–24 h after transfection. Treatment with 3-MA for 18 h beginning 6 h after transfection markedly reduced the levels of N-htt fragments in whole cell lysates and in the detergent insoluble fraction compared to levels of N-htt fragments in untreated cultures (Fig. 3B, left). In the detergent insoluble fraction, the most prominent wt or mutant N-htt fragments found in 3-MA-treated cultures were reduced to 33.8%±7.6 or 56.5%±7.8 of control, respectively (M±SE, P<0.05, one group t-test, n=3). No high molecular weight htt accumulated in the stacking gel (data not shown) of the SDS–PAGE, including the detergent insoluble fraction. This observation counters the possibility that the htt fragments were insoluble and failed to migrate into the gel. Cell viability as assessed by MTT assay did not differ between cultures expressing FH969-100 and treated with or without 3-MA (Fig. 3C) (P>0.05, Student's t-test, n=12). There was no change in levels of endogenous mouse htt with and without 3-MA treatment (small arrow, Fig. 3B). The density of Flag-labeled cells declined with 3-MA treatment to 72% of control for wild-type htt (P<0.003, n=3) and to 60% of control for mutant htt (P<0.001, n=3, data not shown). The levels of intact htt1–969 did not significantly change (95% of control with wt htt and 92% of control with mutant htt, n=3). The marked reduction in N-htt fragments by the autophagy inhibitor 3-MA was unexpected. There are several possible explanations. We showed previously that cell fragments (debris) of clonal striatal cells that have detached from the culture dish in cultures expressing htt1–969 are enriched in caspase cleaved N-htt fragments compared to intact htt (23). The toxicity caused by 3-MA may cause loss of cells that selectively accumulate caspase cleaved N-htt fragments. It is also possible that autophagic pathways may be involved in forming caspase-cleaved N-htt fragments (see results below).

We had shown previously that the production of caspase-cleaved N-htt fragments from htt1–969 in x57 cells coincided with the formation of htt labeled autophagosomes, which fuse and form larger condensed structures in the cytoplasm (htt bodies) that are visible by immunofluorescence (14). In contrast, transient expression of a soluble protein, c-Jun N-terminal protein kinase interacting protein 1 (JIP1), did not produce autophagosome accumulation (14). We compared the effects of 3-MA on the formation of htt bodies detected with anti-Flag antibody. Coincident with the decline in levels of caspase cleaved N-htt fragments in x57 cells, 3-MA treatment reduced the number of cells with Flag labeled bodies formed by htt1–969-18 or htt1–969-100 (Fig. 3D) (Student's t-test, P<0.001, n=6). In another study, we found that there were significantly fewer cells with htt bodies when htt1–969 was expressed in MCF-7 cells, which are deficient in caspase-3 activity (Z.-H. Qin et al., submitted for publication). Collectively, these results suggest that the htt bodies are formed by caspase-cleaved htt fragments and that an autophagic process that generates caspase-cleaved htt is involved in forming htt bodies in vitro.

Previous studies have suggested that N-terminal htt is degraded by the proteasome (18,19). We next studied the relationship between autophagy and proteasome activity, since both constitute means by which cells degrade htt. We immunoprecipitated htt1–969 from cells treated without or with 3-MA (18 h treatment beginning 6 h after transfection) (Fig. 3E) and looked for co-expression of ubiquitin, which binds to proteins removed by the proteasome. Controls that omitted plasmid DNA from the transfection showed no human htt expression (data not shown). There was a substantial increase in ubiquitin immunoreactivity (as indicated by bracket in Fig. 3E) after 3-MA treatment in the htt immunoprecipitates especially high molecular species. The latter may represent htt species that are polyubiquitinated, which is a form of ubiquitin-conjugation recognized by the proteasome. These data suggest that when autophagy is inhibited, more htt is ubiquitinated and may be targeted for removal by the proteasome. An alternative explanation is that 3-MA causes reduced clearance by the proteasome. If this were the case, we would expect to see more robust increases in uncleaved htt after prolonged treatment with 3-MA and we did not. Levels of intact htt were relatively unchanged. These results may also explain why we did not see an accumulation of expressed intact htt after prolonged treatment with 3-MA (Fig. 3B). These data suggest that the two main degradative pathways function co-operatively to degrade htt.

The above results suggested that autophagy may affect caspase-3 activity. We examined the effects of htt expression on levels of active caspase-3 in different cell lines. A 17 kDa active form of caspase-3 can be detected in naive x57 (Fig. 4A) and COS1 (Fig. 4B) cells but not in MCF-7 (data not shown) cells which lack caspase-3. There was a different response of active caspase-3 to mock treatment with the transfection reagent in x57 cells and COS1 cells possibly due to different basal levels of active caspase-3 in the two cell lines. In x57 cells, basal (control) levels of active caspase-3 were high and mock treatment lowered the levels of caspase-3. In COS1 cells, the basal levels of active caspase-3 were low and mock treatment raised the levels of active caspase-3. Exogenous expression of wt and mutant htt1–969 robustly raised levels of active caspase-3 in x57 cells and COS1 cells (Fig. 4A and B) when compared to either the control or mock treatment condition. Treatment with 3-MA for 18 h commencing 6 h after transfection inhibited htt-induced activation of caspase-3 in both cell types (Fig. 4A and B). Thus, inhibiting autophagy with 3-MA lowered levels of active caspase-3. These findings raise the possibility that an autophagic pathway contributes to caspase-cleaved N-htt fragments and htt bodies.



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  		Figure 4. 3-MA blocks htt induced activation of caspase-3. x57 (A) and COS1 (B) cells were transfected with FH969-18 or FH969-100. Cells were incubated with medium with or without 3-MA (10 mM) 6 h after transfection and harvested for western blot analysis 16 h later. Active caspase-3 migrates at a molecular mass of about 17 kDa. The enzyme is increased by htt expression and reduced by 3-MA treatment. Mock condition is transfection reagents without Flag-htt cDNA. (A) and (B) show representative results from three experiments. Control (Cont) is non-transfected cells. Actin levels are relatively unaffected by transfection and treatment with 3-MA. Time line for experimental procedure is the same for (A) and (B).

 
Htt expression upregulates cathepsins ina 3-MA sensitive manner
Lysosomal enzymes, including cathepsin D, accumulate in late autophagosomes during autophagy and degrade proteins delivered to autophagosomes (35). We investigated whether htt accumulation in cells activated cathepsins. In naive x57 cells, we detected cathepsin D as a pro-enzyme (52 kDa) and a partially cleaved intermediate product (46 kDa) (36) but not in an active form (34 kDa) (Fig. 5A). Cathepsin L was expressed as a pro-enzyme of about 30 kDa but not an active form (27 kDa) (Fig. 5B). Exogenous expression of wild-type and mutant htt1–969 increased the levels of the 46 kDa form of cathepsin D and the 30 kDa form of cathepsin L. Treatment of x57 cells with 3-MA for 18 h beginning 6 h after transfection robustly reduced the levels of cathepsin D (46 kDa) and cathepsin L (30 kDa) (Fig. 5A and B). These results show that expression of htt activates cathepsin D and cathepsin L. Furthermore, this activation involves an autophagic mechanism.



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  		Figure 5. 3-MA blocks htt-induced increase in cathepsin D and L. x57 cells were transfected with FH969-18 or FH969-100. Cells were incubated with medium with or without 3-MA (10 mM) 6 h after transfection and then harvested and processed for western blot analysis 16 h later. (A) Cathepsin D (cat D) is expressed as a precursor of about 52 kDa and a processed intermediate of about 46 kDa. Expression of wt or mutant htt increased the levels of the 46 kDa form of cat D. 3-MA treatment blocked the htt-induced increase in the 46 kDa form of cat D. (B) Cathepsin L (cat L) is expressed as a precursor with molecular mass of about 30 kDa. Expression of wt or mutant htt increased the levels of cat L. 3-MA treatment blocked the htt-induced elevation in the 30 kDa form of cat L. Both (A) and (B) show representative results from three experiments. Actin levels are relatively unaffected by htt expression or 3-MA treatment. Time line shows experimental procedure and applies to (A) and (B).

 
Activating autophagy in PC12 cells with inducible htt expression degrades htt
To determine whether autophagy degraded htt in other cell lines expressing lower levels of exogenous htt, we studied PC12 cell lines that had stable ecdysone inducible expression of a cDNA encoding htt1–587 in frame with Flag and GFP tags. Gene expression is induced by stimulating the insect promoter with ponasterone A, a steroid analog of ecdysone. The distribution of Flag-htt-GFP was assessed by analysis of Flag immunocytochemistry or GFP fluorescence. About 80% of PC12-25Q and PC12-144Q cells showed Flag immunoreactivity or GFP immunofluorescence 24–48 h after induction with ponasterone A. The induced Flag-htt-GFP fusion protein distributed diffusely in the cytoplasm with no evidence of accumulation in htt-labeled bodies or inclusions (Fig. 6A). Western blot analysis with anti-htt antibody Ab1 showed that ponasterone A treatment induced expression of a fusion protein with a molecular mass of about 130 kDa in PC12-25Q cells and 140 kDa in PC12-144Q cells (Fig. 6B). Several N-terminal htt fragments also formed.



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  		Figure 6. Effects of 3-MA and lysosomal inhibitors in PC12 cells expressing Flag-htt-GFP (htt1–587). (A) PC12 cell lines were cultured on collagen-coated coverslips and Flag-htt-GFP was induced by ponasterone A (5 µM) for 36 h. Cells were incubated for 6 h with medium containing 10% serum (normal) or 1% (low serum) to induce autophagy, or 1% serum plus 10 mM 3-MA in the continued presence of ponasterone A. Expression and distribution of Flag-htt-GFP was visualized by GFP fluorescence and confocal microscopy. Some cells show reduced GFP fluorescence with low serum. The subcellular distribution of GFP fluorescence in the cytoplasm is not changed by low serum or treatment with 3-MA. (B) and (C) show representative results from two experiments. After a 36 h induction of Flag-htt-GFP expression, cells were incubated for 6 h with regular medium containing 10% serum (+), or 1% serum (-) with or without 10 mM 3-MA, or with or without 10 µM leupeptin, or with or without 10 µM pepstatin A in the continued presence of ponasterone A as indicted. Cells were harvested and processed for western blot analysis with Ab1 antibody. (B) Effects of 3-MA on htt catabolism. Intact (130 kDa for wt htt, indicated by arrowheads) and several large cleaved fragments (indicated by arrows) of inducible Flag-htt-GFP were detected after ponasterone A treatment. Autophagy induced by serum reduction caused a decrease in htt levels and the effects were blocked by 3-MA. (C) Effects of leupeptin and pepstatin A on htt catabolism. Leupeptin and pepstatin A block the decreases in the levels of N-htt fragments induced by serum reduction. The most affected N-htt products are indicated by asterisks. Both (B) and (C) show representative results from two experiments.

 
Serum reduction in undifferentiated PC12 cells induces autophagy (37,38). This response differs from apoptosis, which is triggered by serum withdrawal in differentiated PC12 cells. Reducing serum from 10 to 1% for 6 h had no observable effect on the density or integrity of the induced undifferentiated PC12 cells in culture (Fig. 6A) and active caspase-3 was not detected (data not shown). However, reducing serum decreased the levels of both the intact Flag-htt-GFP and N-htt fragments in the induced PC12 cells. The effects of reducing serum were reversed by 3-MA (Fig. 6B). Treating cells with the lysosome inhibitors pepstatin A or leupeptin partially blocked the effects of serum starvation-induced degradation of N-htt fragments. Pepstatin A, which inhibits the aspartyl protease cathepsin D, was slightly more effective than leupeptin, which inhibits cysteine protease (Fig. 6C).

Cathepsin D (46 kDa) increased 24 h after induction of htt in PC12-25Q cells or PC12-144Q cells (Fig. 7A). In contrast, levels of cathepsin D were unaffected in the control cell line that expressed only Flag-GFP (data not shown). Serum reduction had no additional effect on levels of cathepsin D. 3-MA treatment strongly inhibited the induction of cathepsin D (46 kDa form) by Flag-htt-GFP (Fig. 7A). Similarly, Flag-htt-GFP expression increased the levels of cathepsin L (30 kDa pro-enzyme). 3-MA treatment inhibited the increase in cathepsin L induced by htt expression (Fig. 7B). Consistent with our previous data in x57 cells transiently expressing htt, the results in PC12 cells suggest that htt expression alone (with no additional autophagic stimulation) can upregulate cathepsin D and cathepsin L.



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  		Figure 7. Effects of 3-MA on cathepsin D (A) and L (B) in PC12 cells stably expressing Flag-htt-GFP (1–587 amino acids). Cells were treated as described in Figure 6A and lysates were examined by western blot. (A) Wild-type and mutant htt expression caused an increase in the levels of an intermediate cathepsin D (46 kDa). 3-MA blocked induction of cathepsin D (46 kDa) induced by htt. (B) Expression of htt caused an increase in the levels of cathepsin L (30 kDa form). 3-MA blocked induction of cathepsin L induced by htt. Cathepsin L was less responsive to mutant htt than wt htt. (A) and (B) show representative results from three experiments.

 
Effects of cathepsin D on htt aggregationand degradation
We next studied the role of cathepsin D in htt processing using rodent embryonic tumor cells deficient in cathepsin D (3Y1, cat D-) or overexpressing cathepsin D (4B2 and V2C20, cat D+) cells. Cells were transfected with FH969-18 or FH969-100 and harvested to examine the formation of htt-labeled bodies (accumulations of htt-labeled vacuoles) by immunofluorescence, and the extent of cathepsin D expression and htt processing by western blot analysis. The number of cells expressing htt bodies was significantly greater in cat D- cells than in cat D+ cells (Fig. 8A) (Student's t-test, P<0.001). A precursor (52 kDa), a processed intermediate (46 kDa) and an active form of (34 kDa) cathepsin D were detected in cat D+ cells but not in cat D- cells. The detection of the active 34 kDa fragment in the cat D+ cells is most likely due to the high stable expression of cathepsin D. Levels of the 34 kDa form of cathepsin D increased in cat D+ cells expressing wild-type or mutant htt1–969 (Fig. 8B). In addition, we found that the levels of cleaved N-htt fragments were markedly higher in cat D- cells compared to cat D+ cells (compare lanes 1, 2 and 5, 6 to lanes 3, 4 and 7, 8) (Fig. 8C). There were overall higher levels of mutant N-htt fragments than wt N-htt fragments in cat D+ cells (compare lanes 7 to 3). Treatment of cat D+ cells with pepstatin A increased the levels of N-terminal htt fragments (compare lanes 3 to 4 and lanes 7 to 8), while pepstatin A treatment had no effect in cat D- cells (compare lanes 1 to 2 and lanes 5 to 6). Therefore, cathepsin D is involved in the catabolism of htt fragments that form htt bodies. These results indicated that mutant htt is resistant to cathepsin D-dependent breakdown compared to wt htt.



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  		Figure 8. Analysis of htt bodies, the activation of cathepsin D, htt protein processing in cells expressing human cathepsin D (cat D+) and in cells deficient in cathepsin D (cat D-), and in vitro cleavage of htt by cathepsin D. Cat D- cells (3Y1) and cat D+ cells (4B2 and V2C20) were transfected with FH969-18 or FH969-100 and grown for 24 h. (A) The number of cells with htt bodies is significantly greater in cat D- cells. Cells were processed for immunofluorescence to visualize htt bodies with monoclonal antibody M5 and analyzed as described in Materials and Methods. Statistical analysis was carried out using the Student t-test. ***P<0.001 (comparison between cat D- and cat D+ cells). (B) Western blot analysis of cathepsin D. A precursor (52 kDa), partially processed intermediate (46 kDa) and active (34 kDa) form of cat D were detected in cat D+ cells (4B2) but not in cat D- cells. Htt expression increased levels of an active form of cat D (34 kDa) compared to nontransfected control (Cont) or mock condition (transfecting reagents only). Actin levels are relatively unaffected by treatment conditions. (C) Western blot analysis of htt expression in Cat D- and Cat D+ cells. Sixteen hours after transfection, cells were treated with or without pepstatin A (10 µM) for an additional 8 h. Htt was detected with Ab1. Lower levels of N-htt fragments were detected in cat D+cells (3, 4 and 7, 8) compared to cat D- cells (1, 2 and 5, 6). Pepstatin A treatment had no effect on htt levels in cat D- cells (compare 1 to 2 and 5 to 6) but increased the levels of N-htt fragments in cat D+ cells (compare 3 to 4 and 7 to 8). Higher levels of mutant N-htt fragments than wt N-htt fragment are expressed in Cat-D+ cells (compare 3 to 7). Top panel is a shorter exposure and shows equal levels of uncleaved wild-type and mutant htt (htt1–969) in all conditions. Both (B) and (C) show representative results from two experiments. (D) In vitro degradation of htt by cathepsin D. A western blot is shown of Flag immunoprecipitates from x57 cells expressing FH969-18 or FH969-100 and exposed to different concentrations of cathepsin D alone or with pepstatin A (Pa, 3 nmol). Htt was detected with Ab 1 antibody. Cathepsin D at 0.1 units degraded intact wild-type htt (arrowhead) and htt fragments after 30 min and generated a new htt fragment with molecular mass of about 50 kDa (left arrow). Cathepsin D was much less effective at the same concentration in degrading intact mutant htt and produced a new mutant N-htt mutant htt of about 80 kD (right arrow) that accumulated more than the 50 kD wild-type htt fragment.

 
To further evaluate the sensitivity of wt and mutant htt to cathepsin D, htt1–969 was immunoprecipitated from cell lysates and subjected to degradation by cathepsin D in vitro. Results showed that cathepsin D degraded intact wt htt1–969 and wt N-htt fragments in a dose dependent manner but was less effective under the same treatment conditions in degrading intact mutant htt1–969 and mutant N-htt fragments. Cathepsin D treatment of htt1–969 generated a unique, relatively stable N-htt fragment of about 50 kDa for wt htt and about 80 kDa for mutant htt (Fig. 8D). The mutant 80 kDa N-htt fragment accumulated more than the wt 50 kDa N-htt fragment. Altogether, these results provide additional support that htt is degraded by cathepsin D and that mutant htt is more resistant than wt htt to degradation by cathepsin D, one of the major lysosomal enzymes of the autophagic pathway.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Removal of excess or misfolded proteins is important for maintaining normal cellular function. One pathogenic mechanism in HD may be an inability to process mutant htt and N-terminal mutant htt fragments in HD brain (39). Mutant htt might resist protease activity or be improperly compartmentalized for degradation (36–38). Htt is cleaved by caspases and calpain, which generate stable N-htt fragments in vitro and in brain (23,24,40, Y.J. Kim et al., submitted for publication). The proteasome may contribute to degrading these N-htt fragments (41–45); mutant htt is more resistant than wild-type htt to proteolysis in the HD brain (46). In this study, we demonstrate a role for autophagy and the lysosomal protease cathepsin D in htt catabolism. Moreover, our data suggest that mutant htt resists degradation by cathepsin D compared to wild-type htt. Processing of htt by autophagy and cathepsin D may contribute to HD pathogenesis.

Inhibiting autophagy with 3-MA (clonal striatal cells, PC12 cells) or BAF (clonal striatal cells) increased levels of wild type or mutant htt. Stimulating autophagy with rapamycin (clonal striatal cells) or serum reduction (PC12 cells) degraded htt. In raising intracellular levels of intact htt, 3-MA treatment reduced viability of htt expressing cells. This result differs from the effects of the pan-caspase inhibitor Z-VAD-FMK, which improved the survival of x57 cells expressing htt1–969 by 240–310% (25). The implication is that degradation of htt by autophagy may be important for cell survival. Our findings agree with those of Ravikumar et al. (16,17), who found that mutant htt expressed from exon 1 (htt1–89 with 74Q) was elevated in cells treated with 3-MA or lowered in cells exposed to rapamycin, an activator of autophagy. Inhibiting autophagy with 3-MA increased aggregates formed by mutant htt exon 1 (16) or mutant htt1–287 (this study). We show that products of wt or mutant htt (amino acids 1–287, 1–587, 1–969) that are larger than the protein product of exon 1 are degraded by autophagy. In HD patients, these htt products contain sites of protease cleavage. Protease cleavage generates stable, smaller N-htt fragments, which may aggregate or engage in abnormal protein interactions if the polyglutamine is expanded. At early stages of HD, autophagic activity may help promote the degradation of mutant htt and minimize the production of N-htt fragments. Later in the disease, however, excess mutant htt and autophagic activity may activate caspase-3 and the release of lysosomal proteases, resulting in greater production of N-htt fragments and cellular dysfunction (see below).

Our data suggest that cathepsin D, a major enzyme of autophagosomes, is a critical contributor to htt catabolism by autophagy. Levels of cathepsin D were lower after treating htt expressing cells with 3-MA. At conventional doses of 3-MA used in our studies, 3-MA is a selective class III phosphatidylinositol 3-kinase inhibitor that interferes with the early sequestration phase of autophagy (27,47). It is possible that 3-MA blocked the transport and accumulation of cathepsin D and htt into early autophagosomes in our experiments. We cannot rule out other effects of 3-MA on vesicle trafficking. Endosomes and lysosomes also contain cathepsin D and might participate in htt degradation. In rodent embryonic tumor cells that were devoid of cathepsin D, the degradation of N-htt fragments was slowed as suggested by higher levels of N-htt fragments. Expressing human cathepsin D in this cell line, which originally lacked cathepsin D, produced a rise in an active form of cathepsin D after htt expression. These cells formed fewer htt bodies and had lower levels of N-htt fragments than the parental cells deficient in cathepsin D. Thus, cathepsin D activity modulates the clearance of excess htt in cells. Our in vitro assay confirmed that htt is degraded by cathepsin D and, moreover, that mutant htt is resistant to cathepsin D degradation when compared to wt htt. In both x57 and PC12 cells, upregulation of a pro-form of cathepsin L was also observed; the importance of cathepsin L in htt catabolism is not clear.

Kegel et al. (14) showed cathepsin D immunoreactivity increased more after expression of mutant htt than wild type htt in clonal striatal cells. Mantle et al. (25) found 2- to 3-fold increases in cathepsins D and H in the postmortem striatum of HD patients compared to controls. Increased cathepsins have also been seen in Alzheimer's disease brain (48–50). In the HD brain, excess cathepsin D may cause adverse effects on cell function—the activity of lysosomal proteases might contribute to neurodegeneration (51; see below). In x57 cells and PC12 cells, the active forms of cathepsin D and L were not detected under our experimental conditions. This observation could be due to lower levels of active cathepsin D and L in these cells or to a block in the maturation of cathepsin D and L by htt over expression. The latter seems unlikely. We detected increased levels of active cathepsin D after htt expression in rodent embryonic tumor cells stably expressing human cathepsin D.

Our data suggest that autophagy slows the formation of mutant htt aggregates, by degrading and eliminating small fragments of mutant htt (amino acids 1–287). In the case of large htt products (1–969), autophagy may stimulate caspase-3 dependent cleavage of htt and produce N-htt fragments that accumulate as htt bodies. We found that blocking autophagy with 3-MA for longer periods markedly diminished caspase-3 activity, caspase-cleaved htt products, and the formation of htt autophagic bodies. These results suggest that autophagic pathways may regulate htt degradation, in part by promoting its caspase-dependent cleavage. How autophagy regulates caspase-3 activity is not clear. However, our findings are compatible with data from other investigators showing that some apoptotic stimuli can activate autophagy pathways as well as apoptosis in cells (30,52–54) and that 3-MA treatment in these circumstances can reduce features of apoptosis. 3-MA treatment inhibited cleavage of PARP, a caspase-3 substrate, preserved mitochondrial morphology and prevented release of cytochrome c from mitochondria after apoptotic stimulation (31). Since autophagy may be involved in promoting caspase-dependent htt cleavage and degradation of N-htt fragments, the composite effect of autophagy on cells may depend on the balance between generation and removal of N-htt fragments.

We saw an increase of ubiquitination by immunoprecipitation with an anti-Flag-htt antibody when autophagy was inhibited, supporting the notion that proteasome-ubiquitin pathway plays an important role in degrading htt. Furthermore, these two systems may co-operate to regulate htt catabolism. At early stages of HD, autophagic activity may be beneficial because it degrades mutant htt. When the caspase activity is high and proteasome function is compromised in cells due to mutant htt expression (42,55), overactivation of autophagy may be detrimental because of accumulation of excess caspase-cleaved N-htt fragments.

Furthermore, excessive autophagy may damage cellular organelles, including mitochondria and lysosomes. These events might lead to release of enzymes that cause apoptotic cell death (56–60); namely, lysosomal enzymes released from damaged lysosomes can activate apoptotic cascades (52,54,59). Thus, in advanced HD, an exaggerated autophagic response to accumulation of mutant htt may accelerate cell dysfunction and cell death. Slowing autophagy in later stages of HD and thereby preserving important cellular organelles—and conceivably reducing the formation of N-htt fragments—could provide a novel strategy to treat the disease.

In summary, the present study showed that autophagy plays a role in htt processing and may be important in the removal of mutant htt especially in early stages of HD. Autophagy contributes to the catabolism and cleavage of htt by activating cathepsin D and caspase-3. Mutant N-htt fragments are less efficiently degraded by cathepsin D than are wild type N-htt fragments, thereby contributing to HD pathogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell culture, plasmids and DNA transfection
Mouse clonal striatal cells, x57, were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum, 4.5 g/l glucose and 50 µ/ml penicillin G/streptomycin (25). COS1 cells were cultured in DMEM with 10% fetal bovine serum, 50 units/ml penicillin G/streptomycin, 2 mM glutamine and 1.5 g NaHCO3/l. Rat embryonic tumor cell lines 3Y1 which have a deficiency in cathepsin D (cat D-) were cultured in RPMI1640 containing 5% fetal bovine serum and penicillin G/streptomycin. Three cell lines, 3Y1, V2C20 and 4B2 were kindly provided by Drs H. Rochefort and M. Garcia. 3Y1 cells were stably transfected with human cathepsin D1 or cathepsin D2 to produce V2C20 and 4B2 cells (both cat D+) respectively (60). V2C20 and 4B2 cells were cultured in RPMI1640 containing 10% fetal bovine serum, penicillin G/streptomycin and 0.4 mg/ml G418. Cells were cultured on poly-L-lysine coated coverslips for immunocytochemistry or in 60 mm dishes for immunoblotting. PcDNA3 expression plasmids encoding htt amino acids 1–969 (FH969; based on wt htt with 23 Q) or amino acids 1–287 (FH287) with a Flag tag at the N-terminus and a polyglutamine repeat of 18 or 100 were used in this study. These plasmids were designated as FH969-18, FH969-100, FH287-18 and FH287-100. To disrupt the caspase-3 cleavage sites in htt, we performed site directed mutagenesis in FH969-18 using Gene Editor (Promega) and changed the DNA encoding residues 513 or 552 from aspartic acid to alanine using the following primers to create the mutations: 513D -> A: 5'-ACTCAGTGGCTCGGCCAG-3' and 552D -> A: 5'-ACCTGAATGCTGGGACCCA-3'. All mutations were confirmed by DNA sequencing. At 80% confluence, cells were incubated in 2 ml DMEM containing 5–10 µg of plasmid DNA and 60 µl Superfect (Qiagen, Valencia, CA). After 3 h of incubation, transfection reagents were removed and 6 ml of fresh normal growth medium was added to the dishes and culture was continued to desired time. Cells used for immunocytochemistry were split from the same culture dish into 24-well plates containing poly-L-lysine coated coverslips and culture was continued to desired time. Controls included omission of plasmid DNA in the transfection or DMEM only.

Treatment of cells with 3-methyladenine, bafilomycin A1, pepstatin A, leupeptin or rapamycin
The autophagy inhibitor 3-methyladenine (3-MA, Sigma, St Louis, MO) was freshly dissolved in culture medium 1 h before use at 37°C. The concentration of 3-MA (10 mM) used in this study is the same as that used in studies by other investigators (16,27,30,31) and was not toxic to the cells as determined in preliminary experiments. Wortmannin, another well-known autophagy inhibitor, caused cell death at the concentration used in other studies and could not be used in our experiments. To analyze the effects of 3-MA on htt accumulation, 3-MA was added at 6 or 16 h after transfection of htt cDNA and cells were lysed 14 or 24 h later. Cells were harvested for preparation of whole cell lysates and detergent soluble and insoluble fractions. Bafilomycin A1 (BAF) (Sigma), which inhibits autophagy by blocking the vacuolar ATP pump (61), was used at 200 nM and introduced at 6 h after htt transfection. Cells were harvested at 14 h post-transfection. Pepstatin A (Sigma), which inhibits cathepsin D, and leupeptin (Sigma), which inhibits cathepsin L, were dissolved in sterile PBS and added to culture medium (at 1 : 200 ratio) immediately before use. Six or 16 h after transfection, culture medium was removed and fresh medium was replaced with or without drugs. Rapamycin (Sigma) was dissolved immediately before use in a vehicle containing 90% ethanol and 10% Tween 20 and added to culture medium (0.2 µg/ml) 6 h after transfection; cells were harvested 24 h later. In the analysis of PC 12 cells during serum reduction (serum was reduced from 10 to 1% in medium) and some cells were treated with 3-MA (10 mM), pepstatin A (10 µM) or leupeptin (10 µM) and then harvested and lysed for analysis of inducible htt 6 h after serum reduction. To study the effects of pepstatin A on degradation of htt in cat D- and cat D+ cells, 3Y1 and 4B2 cells were cultured in 60 mm dishes and transfected with FH969-18 or FH969-100. Cells were treated with pepstatin A (10 µM) 16 h after transfection and were harvested for western blot analysis 8 h after pepstatin A treatment.

Immunocytochemistry and quantitative analysis of labeled cells
We have characterized the subcellular localization and toxic effects of human wild-type and mutant htt in clonal striatal cells using biochemical methods and immunofluorescence (14,23,33). Immunofluorescence was performed as described in these publications and Qin et al. (62). Cells (x57) were grown on glass coverlips, fixed in 4% paraformaldehyde and processed for immunocytochemistry 24 h after htt transfection. The total Flag-labeled cells expressing different htt cDNAs, htt aggregates formed by expression of FH287-100 and htt bodies formed by expression of FH969-18 or FH969-100 were detected with anti- Flag (M5, Sigma) monoclonal antibody. Flag-positive x57 cells were counted with epifluorescense microscopy and a 10X objective. Forty microscopic fields were counted per coverslip in cultures expressing FH287-18, FH287-100, FH969-18 and FH969-100 and treated with or without 3-MA. Three coverslips were examined for each condition totaling ~400–2500 cells per group. To quantify the number of cells with htt aggregates and htt bodies, Flag-positive cells were examined with epifluorescense microscopy and a 60x objective. One thousand Flag-labeled cells/coverslip and four or six coverslips were analyzed in each group. Statistical analysis for all quantitative analysis was conducted with the Student's t-test.

Analysis of cell death using MTT assay
x57 cells were treated with (N=12 60 mm culture dishes) or without 3-MA (12 culture dishes) 6 h after transfection of FH969-100 and then analyzed for cell death at 24 h using the method of Mosmann (63). In brief, MTT (Sigma) was added to the media at a final concentration of 125 µg/ml for 1 h at 37° and 5% CO2. The reaction was stopped by adding 300 µl of DMSO and shaking for 20 min. Fifty microliters of sample was transferred to a 96-well micro-titer plate to which 150 µl DMSO was added. One well contained 200 µl of DMSO alone as a blank. The absorbance of the wells was read at 570 nm using a spectrophotometer. The data were statistically analyzed using Student's t-test.

Induction of htt expression in PC12 cells
Pheochromocytoma cells (PC12) were engineered to express an inducible Flag-htt-EGFP with htt amino acids 1–587 and 25Q or 144Q (PC12-25Q and PC12-144Q). The cDNAs were assembled into pIND vector (Invitrogen). The construct encoding htt amino acids 1–587 (based on wt htt with 23 Q, GenBank L12392) had alternating CAG/CAA repeats of either 25Q or 144Q. This encoded htt segment was inserted in frame with Flag sequence at the 5' end and EGFP (enhanced green fluorescent protein, Clontech) sequence at the 3' end. The plasmids were stably transfected into PC12 cell lines as described in Steffan et al. (64) and Peters et al. (65), to generate PC12 cell lines that express the htt gene under the regulation of an ecdysone-sensitive insect promoter. The promoter is repressed in the absence of steroid. The cells were cultured in DMEM containing 10% horse serum, 5% fetal bovine serum, 10 mg/l zeocin, G418 25 mg/l and penicillin G/streptomycin and pH was adjusted to 7.3 with NaHCO3. Expression of PC12-25Q or PC12-144Q was induced with 5 µM ponasterone A, a steroid analog of ecdysone, 2 days after plating cells. Thirty-six hours after induction of htt expression, cells were changed to DMEM containing 1% horse serum (serum deprivation) in the absence or presence of 3-MA (10 mM). The induction of htt and distribution of GFP were examined after cells were cultured on coverslips. Following 6 h of serum deprivation with or without 3-MA (10 mM), cells were fixed in 4% paraformaldehyde in PBS, covered with coverslips, and examined with confocal microscopy (Radiance 2000, Bio-Rad).

Cell fractionation and western blot analysis
For preparation of whole cell lysates, cells were harvested and lysed in buffer containing triethanolamine 10 mM, acetic acid 10 mM, sucrose 250 mM, EDTA 1 mM, DTT 1 mM and protease inhibitors (complete, mini-EDTA free; Bioehringer Mannheim, pH 7.4). Cells were disrupted by passing them through a 26.5 gauge needle 30 times. Cell homogenates generated from this step were used for western blot analysis of htt. The method for preparation of detergent soluble and insoluble fractions was modified from Hazeki et al. (66). Cells were lysed on ice for 30 min in a buffer containing Tris–HCl 50 mM (pH 8.8), NaCl 100 mM, MgCl2 5 mM, Nonidet P-40 0.5%, PMSF 0.5 µg/ml and protease inhibitors (mini-complete). Lysates were centrifuged at 16 000g for 5 min at 4°C. Pellets were resuspended in 100 µl formic acid overnight at 4°C. Samples were then dried under vacuum and treated with 2 µl 2 N NaOH to neutralize pH. Samples were re-suspended in a buffer containing Tris–HCl 20 mM, SDS 2% and DTT 50 mM and sonicated. Protein concentrations in soluble fractions were determined with the BCA kit (Pierce). Protein samples (10–20 µg) were loaded onto 10% acrylamide gels and separated by electrophoresis. After transfer of proteins to nitrocellulose, htt was detected by immunoblotting using the mouse monoclonal antibodies (2166 and 1C2, Chemicon, Temecula, CA) or rabbit polyclonal antibody (Ab1) and enhanced chemiluminesce (ECL). Controls (DMEM or omission of plasmid DNA) show no exogenous human htt on western blot and only endogenous mouse htt as previously described (14). To simplify the presentation, these controls are not shown in the figures here. In western blots, the detection of exogenous human htt after transient transfection in clonal striatal cells or inducible expression in PC12 cells is significantly greater than the endogenous htt in the cells. Some blots were re-probed with anti-actin monoclonal antibody (Sigma). The effects of 3-MA on htt expression were quantitatively analyzed on the films by densitometry using SigmaScan Pro 4 as described previously (57).

The levels of cathepsin D and L and active caspase-3 were analyzed in whole cell lysates by immunoblotting. The following antisera were used: for cathepsin D, rabbit polyclonal antibody (Ab-2, Oncogene) or a goat polyclonal antibody (C-20, Santa Cruz Biotechnology, Santa Cruz, CA); for cathepsin L, goat polyclonal antibody (S-20, Santa Cruz Biotechnology); and for caspase-3, rabbit polyclonal antibody (Asp-175, Cell Signaling Technology, Beverly, MA).

Immunoprecipitation and in vitro degradation of htt
FH969 was expressed in x57 cells for 24 h. Controls included omission of plasmid DNA or no transfection. Cells were lysed in a buffer containing 50 mM Tris (pH 7.4), 250 mM NaCl, 5 mM EDTA and 1% NP-40 and for 10 min on ice. Cells were collected into Eppendorf tubes and centrifuged at 14 000g for 5 min at 4°C. Supernatant was added to 30 µl anti-Flag-agarose (Sigma) and incubated at 4°C for 3 h with shaking. Samples were centrifuged at 14 000g for 5 min and pellets were rinsed five times with the same buffer as described above. Pellets were resuspended in 50 mM Tris-acetate buffer (pH 3.3) and boiled for 5 min. Fifteen microliters of the resuspended solution from each tube was added to 5 µl enzyme solution containing cathepsin D 0, 0.1, 0.05, 0.025 or 0.1 units cathepsin D plus 3 nmol pepstatin A. Samples were incubated at 37°C for 30 min. The reaction was terminated by adding 5 µl loading buffer, 1 µl 6.7 N NaOH to neutralize pH and boiled for 5 min. Samples were subjected to electrophoresis and htt fragments were detected with Ab 1 rabbit antibody. Some cells were treated with 10 mM 3-MA 6 h after htt expression and immunoprecipitation was performed with Flag agarose as described above. Two sets of samples were separated in the same SDS–PAGE and transferred to nitrocellulose. One set was examined for ubiquitin immunoreactivity (Sigma) and the other for htt immunoreactivity with antibody Ab1.


   ACKNOWLEDGEMENTS
 
This work was supported by NIH Grants NS16367 and NS13579 to M.D., NS38194 to N.A., a grant from the HDSA to M.D., HDF Cure HD Initiative to L.M.T. and HDSA Coalition for the Cure grant to L.M.T.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +1 6172765762; Email: difiglia{at}helix.mgh.harvard.edu Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Hershko, A. and Ciechanover, A. (1982) Mechanisms of intracellular protein breakdown. Annu. Rev. Biochem., 51, 335–364.[CrossRef][Web of Science][Medline]

  2. Voges, D., Zwickl, P. and Baumeister, W. (1999) The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu. Rev. Biochem., 68, 1015–1068.[CrossRef][Web of Science][Medline]

  3. Beynon, R.J. and Bond, J.S. (1986) Catabolism of intracellular protein: molecular aspects. Am. J. Physiol., 251 (2 Pt 1), C141–152.[Medline]

  4. Kim, J. and Klionsky, D.J. (2000) Autophagy, cytoplasm-to-vacuole targeting pathway, and pexophagy in yeast and mammalian cells. Annu. Rev. Biochem., 69, 303–342.[CrossRef][Web of Science][Medline]

  5. Klionsky, D.J. and Emr, S.D. (2000) Autophagy as a regulated pathway of cellular degradation. Science, 290, 1717–1721.[Abstract/Free Full Text]

  6. Punnonen, E.T., Autio, S., Kaija, H. and Reunanen, H. (1993) Autophagic vacuoles fuse with the prelysosomal compartment in cultured rat fibroblasts. Eur. J. Cell. Biol., 61, 54–66.[Web of Science][Medline]

  7. Glaumann, H., Ericsson, J.L. and Marzella, L. (1981) Mechanisms of intralysosomal degradation with special reference to autophagocytosis and heterophagocytosis of cell organelles. Intl. Rev. Cytol., 73, 149–182.

  8. Teckman, J.H. and Perlmutter, D.H. (2000) Retention of mutant {alpha}1-antitrypsin Z in endoplasmic reticulum is associated with an autophagic response. Am. J. Physiol. Gastrointest. Physiol., 279, G961–G974.[Abstract/Free Full Text]

  9. Jeffrey, M., Scott, J.R., Williams, A. and Fraser, H. (1992) Ultrastructural features of spongiform encephalopathy transmitted to mice from three species of bovidae. Acta Neuropathol. (Berl), 84, 559–569.[CrossRef][Medline]

  10. Anglade, P., Vyas, S., Javoy-Agid, F., Herrero, M.T., Michel, P.P., Marqurez, J., Mouatt-Prigent, A., Ruberg, M., Hirsch, E.C. and Agid, Y. (1997) Apoptosis and autophagy in nigral neurons of patients with Parkinson's disease. Histol. Histopathol., 12, 25–31.[Web of Science][Medline]

  11. Hirai, K., Aliev, G., Nunomura, A., Fujioka, H., Russell, R.L., Atwood, C.S., Johnson, A.B., Kress, Y., Vinters, H.V., Tabaton, M. et al. (2001) Mitochondrial abnormalities in Alzheimer's disease. J. Neurosci., 21, 3017–3023.[Abstract/Free Full Text]

  12. Stefanis, L., Larsen, K.E., Rideout, H.J., Sulzer, D. and Greene, L.A. (2001) Expression of A53T mutant but not wild-type alpha-synuclein in PC12 cells induces alterations of the ubiquitin-dependent degradation system, loss of dopamine release, and autophagic cell death. J. Neurosci., 21, 9549–9560.[Abstract/Free Full Text]

  13. Uchiyama, Y. (2001) Autophagic cell death and its execution by lysosomal cathepsins. Arch. Histol. Cytol., 64, 233–246.[CrossRef][Web of Science][Medline]

  14. Kegel, K.B., Kim, M., Sapp, E., McIntyre, C., Castaño, J.G., Aronin, N. and DiFiglia, M. (2000) Huntingtin expression stimulates endosomal-lysosomal activity, endosome tubulation, and autophagy. J. Neurosci., 20, 7268–7278.[Abstract/Free Full Text]

  15. Petersén, Å., Larsen, K.E., Behr, G.G., Romero, N., Przedborski, S., Brundin, P. and Sulzer, D. (2001) Expanded CAG repeats in exon 1 of the Huntington's disease gene stimulate dopamine-mediated striatal neuron autophagy and degeneration. Hum. Mol. Genet., 10, 1243–1254.[Abstract/Free Full Text]

  16. Ravikumar, B., Duden, R. and Rubinsztein, D.C. (2002) Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. Hum. Mol. Genet., 11, 1107–1117.[Abstract/Free Full Text]

  17. Ravikumar, B., Stewart, A., Kita, H., Kato, K., Duden, R. and Rubinsztein, D.C. (2003) Raised intracellular glucose concentrations reduced aggregation and cell death caused by mutant huntingtin exon 1 by decreasing mTOR phosphorylation and inducing autophagy. Hum. Mol. Genet., 12, 985–994.[Abstract/Free Full Text]

  18. DiFiglia, M., Sapp, E., Chase, K.O., Davies, S.W., Bates, G.P., Vonsattel, J.P. and Aronin, N. (1997) Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science, 277, 1990–1993.[Abstract/Free Full Text]

  19. Davies, S.W., Turmaine, M., Cozens, B.A., DiFiglia, M., Sharp, A.H., Ross, C.A., Scherzinger, E., Wanker, E.E., Mangiarini, L. and Bates, G.P. (1997) Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell, 90, 537–548.[CrossRef][Web of Science][Medline]

  20. Moulder, K.L., Onodera, O., Burke, J.R., Strittmatter, W.J. and Johnson, E.M., Jr (1999) Generation of neuronal intranuclear inclusions by polyglutamine-GFP: analysis of inclusion clearance and toxicity as a function of polyglutamine length. J. Neurosci., 19, 705–715.[Abstract/Free Full Text]

  21. Li, H., Li, S.-H., Yu, Z.-X., Shelbourne, P. and Li, X.-J. (2001) Huntingtin aggregate-associated axonal degeneration is an early pathological event in Huntington's disease mice. J. Neurosci., 21, 8473–8481.[Abstract/Free Full Text]

  22. Wellington, C.L., Singaraja, R., Ellerby, L.M., Savill, J., Roy, S., Leavitt, B., Cattaneo, E., Hackam, A., Sharp, A., Thornberry, N. et al. (2000) Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and nonneuronal cells. J. Biol. Chem., 275, 19831–19838.[Abstract/Free Full Text]

  23. Kim, Y.J., Yi, Y., Sapp, E., Wang, Y., Cuiffo, B., Kegel, K.B., Qin, Z.-H. and Aronin, N. (2001) Caspase 3-cleaved N-terminal fragments of wild-type and mutant huntingtin are present in normal and Huntington's disease brains, associate with membranes, and undergo calpain-dependent proteolysis. Proc. Natl Acad. Sci. USA, 98, 12784–12789.[Abstract/Free Full Text]

  24. Gafni, J. and Ellerby, L.M. (2002) Calpain activation in Huntington's disease. J. Neurosci., 22, 4842–4849.[Abstract/Free Full Text]

  25. Mantle, D., Falkous, G., Ishiura, S., Perry, R.H. and Perry, E.K. (1995) Comparison of cathepsin protease activities in brain tissue from normal cases and cases with Alzheimer's disease, Lewy body dementia, Parkinson's disease and Huntington's disease. J. Neurol. Sci., 131, 65–70.[CrossRef][Web of Science][Medline]

  26. Blommaart, E.F., Krause, U., Schellens, J.P., Vreeling-Sindelarova, H. and Meijer, A.J. (1997) The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur. J. Biochem., 243, 240–246.[Web of Science][Medline]

  27. Petiot, A., Ogier-Denis, E., Blommaart, E.F.C., Meijer, A.J. and Codogno, P. (2000) Distinct classes of phosphatidylinositol 3'-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J. Biol. Chem., 275, 992–998.[Abstract/Free Full Text]

  28. Schu, P.V., Takegawa, K., Fry, M.M., Stack, J.H., Waterfield, M.D. and Emr, S.D. (1993) Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science, 260, 88–91.[Abstract/Free Full Text]

  29. Futter, C.E., Collinson, L.M., Backer, J.M. and Hopkins, C.R. (2001) Human VPS34 is required for internal vesicle formation within multivesicular endosomes. J. Cell Biol., 155, 1251–1264.[Abstract/Free Full Text]

  30. Xue, L., Fletcher, G.C. and Tolkovsky, A.M. (1999) Autophagy is activated by apoptotic signaling in sympathetic neurons: an alternative mechanism of death execution. Mol. Cell. Neurosci., 14, 180–198.[CrossRef][Web of Science][Medline]

  31. Xu, L., Borutaite, V. and Tolkovsky, A.M. (2002) Inhibition of mitochondrial permeability transition and release of cytochrome c by anti-apoptotic nucleoside analogues. Biochem. Pharmacol., 64, 441–449.[CrossRef][Web of Science][Medline]

  32. Yamamoto, A., Tagawa, Y., Yoshimori, T., Moriyama, Y., Masaki, R. and Tashiro, Y. (1998) Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct. Funct., 23, 33–42.[Web of Science][Medline]

  33. Kim, M., Lee, H.-S., LaForet, G., McIntyre, C., Martin, EL., Chang, P., Kim, T.W., William, M., Reddy, P.H., Tagle, D. et al. (1999) Mutant huntingtin expression in clonal striatal cells: dissociation of inclusion formation and neuronal survival by caspase inhibition. J. Neurosci., 19, 964–973.[Abstract/Free Full Text]

  34. Raught, B., Gingras, A.-C. and Sonnenberg, N. (2001) The target of rapamycin (TOR) proteins. Proc. Natl Acad. Sci. USA, 98, 7037–7044.[Abstract/Free Full Text]

  35. Dunn, W.A., Jr (1990) Studies on the mechanisms of autophagy: maturation of the autophagic vacuoles. J. Cell. Biol., 110, 1935–1945.[Abstract/Free Full Text]

  36. Barrera, C.A., Ye, G., Espejo. R., Gunasena, S., Almanza, R., Leary, J.F., Crowe, S.E., Ernst, P.B. and Reyes, V.E. (2001) Expression of cathepsin B, L, S, and D by gastric epithelial cells implicates them as antigen presenting cells in local immune responses. Hum. Immunol., 62, 1081–1091.[CrossRef][Web of Science][Medline]

  37. Mortimore, G.E. and Poso, A.R. (1987) Intracellular protein catabolism and its control during nutrient deprivation and supply. Annu. Rev. Nutr., 7, 539–564.[CrossRef][Web of Science][Medline]

  38. Ohsawa, Y., Isahara, K., Kanamori, S., Shibata, M., Kametaka, S., Gotow, T., Watanami, E. and Uchiyama, Y. (1998) An ultrastructural and immunohistochemical study of PC12 cells during apoptosis induced by serum deprivation with special reference to autophagy and lysosomal cathepsins. Arch. Histol. Cytol., 61, 395–403.[Web of Science][Medline]

  39. Mende-Mueller, L.M., Toneff, T., Hwang, S.R., Chesselet, M.F. and Hook, V.Y. (2001) Tissue-specific proteolysis of huntingtin (htt) in human brain: evidence of enhanced levels of N- and C-terminal htt fragments in Huntington's disease striatum. J. Neurosci., 21, 1830–1837.[Abstract/Free Full Text]

  40. Wellington, C.L., Ellerby, L.M., Hachman, A.S., Margolis, R.L., Trifiro, M.A., Singaraja, R., McCutcheon, K., Salvesen, G.S., Propp, S.S., Bromm, M. et al. (1998) Caspase cleavage of gene products associated with triple expansion disorders generates truncated fragments containing the polyglutamine tract. J. Biol. Chem., 273, 9158–9167.[Abstract/Free Full Text]

  41. Wyttenbach, A., Carmichael, J., Swartz, J., Furlong, R.A., Narain, Y., Rankin, J. and Rubinsztein, D.C. (2000) Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington's disease. Proc. Natl Acad. Sci. USA, 97, 2898–2903.[Abstract/Free Full Text]

  42. Bence, N.F., Sampat, R.M. and Kopito, R.R. (2001) Impairment of the ubiquitin-proteasome system by protein aggregation. Science, 292, 1552–1555.[Abstract/Free Full Text]

  43. Jana, N.R., Zemskov, E.A., Wang, G.H. and Nukina, N. (2001) Altered proteasomal function due to the expression of polyglutamine-expanded truncated N-terminal huntingtin induces apoptosis by caspase activation through mitochondrial cytochrome c release. Hum. Mol. Genet., 10, 1049–1059.[Abstract/Free Full Text]

  44. Martin-Aparicio, E., Yamamoto, A., Hernández, F., Hen, R., Avila, J. and Lucas, J.J. (2001) Proteasomal-dependent aggregate reversal and absence of cell death in a conditional mouse model of Huntington's disease. J. Neurosci., 21, 8772–8781.[Abstract/Free Full Text]

  45. Lunkes, A., Lindenberg, K.S., Ben-Haiem, L., Weber, C., Devys, D., Landwehrmeyer, G.B., Mandel, J.L. and Trottier, Y. (2002) Proteases acting on mutant huntingtin generate cleaved products that differentially build up cytoplasmic and nuclear inclusions. Mol. Cell, 10, 259–269.[CrossRef][Web of Science][Medline]

  46. Dyer, R.B. and McMurray, C.T. (2001) Mutant protein in Huntington disease is resistant to proteolysis in affected brain. Nat. Genet., 29, 270–278.[CrossRef][Web of Science][Medline]

  47. Seglen, P.O. and Gorden, P.B. (1982) 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc. Natl Acad. Sci. USA, 79, 1889–1892.[Abstract/Free Full Text]

  48. Cataldo, A.M. and Nixon, R.A. (1990) Enzymatically active lysosomal proteases are associated with amyloid deposits in Alzheimer brain. Proc. Natl Acad. Sci. USA, 87, 3861–3865.[Abstract/Free Full Text]

  49. Cataldo, A.M., Barnett, J.L., Berman, S.A., Li, J., Quarless, S., Bursztajin, S., Lippa, C. and Nixon, R.A. (1995) Gene expression and cellular content of cathepsin D in Alzheimer's disease brain: evidence for early up-regulation of the endosomal-lysosomal system. Neuron, 14, 671–680.[CrossRef][Web of Science][Medline]

  50. Hetman, M., Filipkowski, R.K., Domagala, W. and Kaczmarek, L. (1995) Elevated cathepsin D expression in kainate-evoked rat brain neurodegeneration. Exp. Neurol., 136, 53–63.[CrossRef][Web of Science][Medline]

  51. Bahr, B.A. and Bendiske, J. (2002) The neuropathogenic contributions of lysosomal dysfunction. J. Neurochem., 83, 481–489.[CrossRef][Web of Science][Medline]

  52. Isahara, K., Ohsawa, Y., Kanamori, S., Shibata, M., Waguri, S., Sato, N., Gotow, T., Watanabe, T., Momoi, T., Urase, K. and Kominami, E. (1999) Regulation of a novel pathway for cell death by lysosomal aspartic and cysteine proteases. Neuroscience, 91, 233–249.[CrossRef][Web of Science][Medline]

  53. Bursch, W., Ellinger, A., Gerner, C., Frohwein, U. and Schulte-Hermann, R. (2000) Programmed cell death (PCD), Apoptosis, autophagic PCD, or both? Ann. NY Acad. Sci., 926, 1–12.[CrossRef][Medline]

  54. Roberg, K., Kågedal, K. and Õllinger, K. (2002). Microinjection of cathepsin D induces caspase-dependent apoptosis in fibrolasts. Am. J. Pathol., 161, 89–96.[Abstract/Free Full Text]

  55. Miyashita, T., Matsui, J., Ohtsuka, Y., Mami, U., Fujishima, S., Okamura-Oho, Y., Inoue, T. and Yamada, M. (1999) Expression of extended polyglutamine sequentially activates initiator and effector caspases. Biochem. Biophys. Res. Comm., 257, 724–730.[CrossRef][Web of Science][Medline]

  56. Knechi, E., Martinez-Ramón, A. and Grisolia, S. (1988) Autophagy of mitochondria in rat liver assessed by immunogold procedures. J. Histochem. Cytochem., 36, 1433–1440.[Abstract]

  57. Elmore, S.P., Qian, T., Grisson, S.F. and Lemasters, J.J. (2001) The mitochondrial permeability transition initiates autophagy in rat hepatocytes. FASEB J., 15, 2287–2287.

  58. Larsen, K.E., Fon, E.A., Hastings, T.G., Edwards, R.H. and Sulzer, D. (2002) Methamphetamine-induced degeneration of dopaminergic neurons involves autophagy and upregulation of dopamine synthesis. J. Neurosci., 22, 8951–8960.[Abstract/Free Full Text]

  59. Stoka, V, Turk, B., Sehendel, SL., Kim, T.-H., Cirman, T., Snipas, S.J., Ellerby, L.M., Bredesen, D., Freeze, H., Abrahamson, M. et al. (2001) Lysosomal protease pathways to apoptosis: cleavage of Bid, not pro-caspases, is the most likely route. J. Biol. Chem., 276, 3149–3157.[Abstract/Free Full Text]

  60. Liaudet, E., Derocq, D., Rochefort, H. and Garcia, M. (1995) Transfected cathepsin D stimulates high density cancer cell growth by inactivating secreted growth inhibitors. Cell Growth Diff., 6, 1045–1052.[Abstract]

  61. Yoshimori, T., Yamamoto, A., Moriyama, Y., futai, M. and Tashiro, Y. (1991) Bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J. Biol Chem., 266, 17707–17712.[Abstract/Free Full Text]

  62. Qin, Z.-H., Wang, Y., Kikly, K.K., Sapp, E., Kegel, K.B., Aronin, N. and DiFiglia, M. (2001) Pro-caspase-8 is predominantly localized in mitochondria and released into cytoplasm upon apoptotic stimulation. J. Biol. Chem., 276, 8079–8086.[Abstract/Free Full Text]

  63. Mosmann, T.T. (1983) Rapid colourmetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol Meth., 65, 55–63.[CrossRef][Web of Science][Medline]

  64. Steffan, J.S., Bodai, L., Pallos, J., Poelman, M., McCampbell, A., Apostol, B.L., Kazantsey, A., Schmidt, E., Zhu, Y.Z., Greenwald, M. et al. (2001) Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila. Nature, 413, 739–743.[CrossRef][Medline]

  65. Peters, P.J., Ning, K., Palacios, F., Boshans, R.L., Kazantsev, A., Thompson, L.M., Woodman, B., Bates, G.P. and D'Souza-Schorey, C. (2002) Arfaptin 2 regulates the aggregation of mutant huntingtin protein. Nat. Cell Biol., 4, 240–245.[CrossRef][Web of Science][Medline]

  66. Hazeki, N., Tukamoto, T., Goto, J. and Kanazawa, I. (2000) Formic acid dissolves aggregates of an N-terminal huntingtin fragment containing an expanded polyglutamine tract: applying to quantification of protein components of the aggregates. Biochem. Biophy. Res. Comm., 277, 386–393.[CrossRef][Web of Science][Medline]


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