Association Of Proteasome Activity With Accumulation And Cytotoxicity Of Mutant Huntingtin Aggregates

Huntingdon disease (HD) is an autosomal dominant inherited neurodegenerative disease caused by the excess expansion of trinucleotide repeat (CAG), coding an N terminus protein sequence (polyQ) of huntintin (htt). The representative pathological feature of HD is the formation of cytoplasmic mutant htt aggregates. Normal htt is diffused in the soma, while mutant htt in HD patients or animal model is located at nuclear and process, including axon and axon terminus, of neurons as insoluble aggregates which is called intranuclear aggregates and neuropil aggregates respectively. It is unclear whether these insoluble aggregates are related to the neurodegeneration in HD or the aggregates isolate the proteins with potential toxicity.Ubiquitin-Proteasome system (UPS) is one of the intracellular protease systems. UPS tags misfolded proteins for refolding or degradation and hereby protects cells from the toxic effect of aggregated proteins. If UPS function is impaired, proteins will not able to fold correctly and these misfolded proteins will accumulate to be aggregates. Due to the malfunction of UPS, the aggregates can not be degraded promptly and gather to be a large amount. It is reported that the accumulation of aggregates is associated with the hypofunction of UPS. So this hypofunction may be one of the mechanisms of HD. However, it has not been clear up till now the relationship among mutant protein aggregation, UPS hypofunction, and neurodegeneration.Reactive oxidative species (ROS) upgrade in neurodegeneration. Oxidative stress is involved in HD pathophysiology and may influence UPS function. But it remains unsolved whether the cytotoxicity of mutant htt is caused by UPS impairment through oxidative stress.In this paper, we proved that the inhibition of proteasome activity enhances the mutant htt aggregation, on the basis of which we further evidenced the cytotoxicity of mutant htt, the effects of mutant htt on the activity of proteasome, and the effects of oxidative stress in proteasome impairment process.1. The Effect of inhibiting proteasome activity on Formation of mutant htt aggregates We first established stable cell lines that express amino-terminal fragments of normal (20Q) or mutant Htt (120Q) by pEGFP-htt-20Q or pEGFP-htt-120Q transfection and G418 screening. We found 20Q htt diffused in the cytoplasm and did not or occasionally form aggregation but 120Q formed cytoplasmic aggregates in some cells. Using N-Acetyl-Leu-Leu-Norleu-al (ALLN), a proteasome inhibitor, under a fluorescent microscope, we found 20Q cell lines did not exhibit aggregates but cells that emerged aggregation in 120Q cell lines showed a time and dose dependent increase. In 120Q cell lines, the aggregated htt increased in a time and ALLN dose dependent in manner in western blotting. It is suggested that the formation of mutant htt aggregates was related to the downgrade of proteasome activity.To examine the activity of proteasome directly and dynamically, pEGFP-CL1 and pDsRed-CL1, containing degradation signal of proteasome CL1 and, were transfected into HEK-293 cell line and established stable monoclonal cell lines: GFPu and DsRed2u. Treated by ALLN, changes of GFP and DsRen were detected in these cell lines under a laser confocal microscope and by western blotting. Fluorescent intensity in GFPu and DsRed2u incresase time and ALLN dose dependently. Fluorescin in these cell lines increased too. It was suggested that GFPu and DsRedu was an efficient dynamic system to directly observe proteasome activity. The fluorescent intensity and fluoresin content represented the activity of proteasome indirectly and correlated negatively.To further illuminate the relationship between the changes of proteasome activity and that of aggregates, we transfected pDsRed2-20Q and pDsRed2-120Q into GFPu cell line repectively, and treated it with ALLN. Western Blotting indicated that the amount of aggregates in 120Q cell line and GFP expression had positive correlation. So did that and GFP fluorescence intensity, elucidating that the hypofunction of proteasome induced the formation of aggregation by mutant htt.2. The effect of Inhibiting proteasome activity on cytotoxicity of mutant huntingtinMTT assay demonstrated an ALLN time and dose dependent decrease in cell vigor. The decrease was more significant in 150Q than 20Q.We transfected pEGFP-Htt-20Q and pEGFP-Htt-120Q into a Hela cell line that expressed Caspase 3 Fret probe. It is indicated that FRET obviously declined in cells with 150Q-htt aggregation but not that much in cells with diffused 150Q-htt; and yet in 20Q cells FRET was not disturbed. So mutant htt could activate caspase-3 and induced apoptosis.Treated with ALLN, caspase-3 probe transfected 20Q cells exhibit slightly decrease in FRET phenomenon but 120Q cells obviously declineAfter GFPu cell was co-transfected by caspase-3 and pDsRed2-Htt-20Q or pDsRed2-Htt-120Q and then treated with ALLN, 120Q cell showed more aggregates, enhanced GFPu fluorescent intensity, decreased Fret phenomenon. In 20Q cells, although GFPu was more intense but FRET phenomenon only slightly decrease. It was suggested that proteasome inhibitor advanced the mutant htt aggregation dependent cytotoxicity, including apoptosis.3. The role of oxidative stress in the influence of mutant huntingtin on proteasome activityTo elucidate the Effects of mutant htt on proteasome activity, we transfected pDsRed2-Htt-20Q and pDsRed2-Htt-120Q into GFPu cell line and observed under a laser confocal microsope or analysed by using western blotting. Cells with 120Q-htt, especially those with aggregates, showed more intensive fluorescence, but 20Q-htt can not affect fluorescent intensity and protein level.Using western blotting and laser confocal microscope technique, GFP level in H2O2 treated GFPu increased significantly. We transfected pDsRed2-htt-20Q and pDsRed2-htt-120Q into GFPu stable cell line and treated them with H2O2, H2O2+Edaravone, or Edaravone. It is indicated that Edaravone reduced the GFPu enhancement and decline the aggregates.After pDsRed2-Htt-20Q and pDsRed2-Htt-120Q transfected GFPu cell lines had been incubation with H2O2, H2O2+Edaravone, or Edaravone for 24h, MTT assay showed that H2O2+Edaravone group indicated higher cell vigor than 0.1mM H2O2 group, and Edaravone group higher than DMEM group, especially in 120Q cell lines.We then transfected pEGFP-htt-20Q and pEGFP-htt-120Q to HELA cells that express Caspase-3 FRET probe. After 24h's incubation with H2O2, H2O2+Edaravone, or Edaravone. The TRET detection in HELA cell showed a decreased FRET occurrence in H2O2 group. However treated with H2O2 + Edaravone, FRET occurrence significantly increased. With Edaravone treatment FRET occurrence increased in pDsRed2-Htt-120Q transfected cellsIt is suggested that mutant htt inhibit proteasome function or impaired its activity through oxidative stress and hereby reduced the degradation of htt aggregates, which eventually caused cytotoxicity, including apoptosis.Conclusion: Mutant htt inhibit proteasome function or impaired its activity through oxidative stress. Hence, mutant htt in neuron cannot be eliminated and led to neurodegeneration. This may be one of the important mechanisms in HD pathogeny. Free radical scavenger such as Edaravone protected proteasome activity and function and may be have therapeutic action.