Analysis For The Effect Of Mutant Huntingtin On Cleavage Of CREST By Calpain-2 And Its Cytotoxicity

Background Huntington’s disease（HD）is a hereditary neurodegenerative disease caused by HD gene mutation.The mutant HD gene encodes a mutant huntingtin（mhtt）with an abnormally extended polyglutamine（poly-Q）structure（Q repeat number > 35）at the amino terminal.Although a large number of studies have shown that mHtt can produce cytotoxicity and cause neurodegeneration through a variety of ways,so far,the pathogenesis of HD has not been fully revealed,and there is no effective formula for the treatment of HD.Cell cycle re-entry of the neurons exists in a variety of neurodegenerative diseases such as HD.It is characterized by the phosphorylation of retinoblastoma protein（Rb）and the abnormal up-regulation of cyclins and cyclin dependent kinases（CDKs）in mature neurons.Previous studies of our laboratory found that mHtt could down-regulate the level of full-length calcium responsive transactor（CREST）and produce short CREST fragments.This effect of mHtt could be blocked by the inhibitor of calpain-1 / 2,and overexpression of CREST could reduce the cytotoxicity of mHtt,it was therefore suggested that mHtt might inhibit the role of CREST in stabilizing cell cycle and promoting neuronal differentiation by increasing the enzymatic cleavage of CREST by calpain-1 / 2.Further,mass spectrometry analysis and coimmunoprecipitation（Co-IP）assay confirmed that calpain-2 rather than calpain-1 had specific enzymatic digestion effect on CREST.It has been reported that calpain-1,2,5,7 and 10 are activated in the brains of HD patients;however,it is unclear whether calpain-5,7 and 10 also have effective enzymatic digestion effect on CREST.Objective To clarify whether mHtt promotes the enzymatic degradation of CREST that plays a regulatory role in neuronal differentiation by affecting the expressions of calpains,and then interfering with the cell cycle of neurons to produce cytotoxicity,so as to provide a scientific basis for further revealing the pathogenesis of HD and establishing the application of the calpain cleavage-site blocking peptides to prevent and treat HD neurodegeneration,this study will first prove the types of calpains whose expressions are affected by mHtt,and then analyze the types of caplains that can cleave CREST and the influence of mHtt on cleavage of CREST by calpains.Furthermore,we plan to analyze the changes of mHtt-induced cytotoxicity and cell cycle re-entry after blocking the specific calpain-2 cleavage on CRREST by using the competitive enzyme cleavage site blocking peptides that are designed and synthesized according to the cleavage site of CREST by calpain-2.Methods（1）After transfection and expression of normal Htt（Htt-20Q）or mHtt（Htt-160Q）in N2 a cells,semi-quantitative RT-PCR and q RT-PCR were used to detect the effect of mHtt on the m RNA expression levels of calpain-1,2,5,7,10.（2）calpain-2,5,7 or 10 fused with 3×Flag were transfected and expressed in N2 a cells,respectively,purified by immunoprecipitation with anti-Flag magnetic beads,incubated with prokaryotic expressed and purified full-length GFP-CREST in corresponding calpain enzyme digestion system,and the enzyme digestion effect of each calpain on CREST（whether the level of full-length CREST decreased and whether truncated CREST fragments appeared）was detected by Western blot.In N2 a cells transiently expressing GFP-Htt-20 Q or GFP-Htt-160 Q,the synthetic si RNA of calpain-2,5,7 or 10 was cotransfected,respectively,and Western blot was used to detect the type（s）of calpain（s）through which mHtt induce cleavage of endogenous CREST.（3）The purified 3×Flagcalpain-2 that was transfected and expressed in HEK293 T cells was incubated with the purified His-CREST and purified His-3×Flag-Htt-20 Q or 40 Q,which were expressed in prokaryotic cells,the effect of mHtt on the interaction between calpain-2 and CREST was detected by vitro magnetic beads Pull-down assay,and whether mHtt directly promotes the enzymatic cleavage of calpain-2 on CREST was detected by Western blotting.（4）The competitive Tat transmembrane blocking peptide was designed and made according to the two sites of CREST digested by calpain-2,and added into N2 a cells transfected with Htt-20 Q or 160 Q.The microscope was used to detect whether the transmembrane blocking peptides could reduce the inhibition of mHtt on the neurite growth of N2 a cells,Trypan blue staining was used to detect whether the transmembrane blocking peptides could inhibit the promotion of mHtt on the death of N2 a cells,and Western blot was used to detect whether the transmembrane blocking peptides could block the degradation and the expression of neuronal differentiation markers and the expression of neuronal cell cycle regulatory protein.（5）Full-length CREST and different calpain-2-cleaved fragments of CREST were transfected and expressed in N2 a cells,respectively,the localization and distribution of calpain-2-cleaved fragments of CREST were detected by immunofluorescence staining,Trypan blue staining and Caspase-3 Western blotting were used to detect whether calpain-2-cleaved fragments of CREST were cytotoxic.Results（1）mHtt up-regulates expression of calpain-1,2,5,7,10 m RNA: semi-quantitative RT-PCR and real time RT-PCR showed that the m RNA expression levels of calpain-1,2,5,7 and 10 in N2 a cells transfected with Htt-160 Q were significantly higher than those in N2 a cells transfected with Htt-20 Q,indicating that mHtt can affect its activity by up-regulating the expression of calpain-1,2,5,7 and 10.（2）mHtt promotes the enzymatic Cleavage of CREST by calpain-2,5,7 and 10: Western blot analysis showed that after the purified CREST was treated with purified calpain-2,5,7 and 10 in the corresponding calpain enzyme digestion system,the fulllength CREST level of the 4 kinds of calpain could be significantly decreased.Among them,calpain-2 could significantly increase the level of 35 k D CREST fragment,and calpain-5,7 and 10 could significantly increase the level of 13 k D CREST fragment.It is proved that not only calpain-2 can cleave CREST to produce CREST fragments,but also calpain-5,7 and 10 can cleave and degrade CREST to produce smaller CREST fragments.Furthermore,the full-length CREST level in N2 a cells transfected with Htt-160 Q was significantly lower than that of N2 a cells transfected with Htt-20Q;when calpain-2,5,7 or 10 were silenced with corresponding si RNA,the effect of Htt-160 Q on reducing the level of full-length CREST was significantly weakened,and the effect of silencing calpain-2 on inhibiting mHtt and increasing the cleavage fragment of CREST（35 k D） was the most obvious.This further provides experimental evidence that mHtt may promote the enzymatic degradation of CREST by up-regulating the expression or increasing the activity of calpain-2 and other calpains.（3）mHtt directly promotes the enzymatic degradation of CREST by calpain-2 through enhancing the interaction between calpain-2 and CREST: Vitro magnetic beads Pull down analysis showed that purified Htt-160 Q could significantly enhance the interaction between purified calpain-2 and CREST,compared with purified Htt-20Q;In calpain-2 enzyme digestion system,the addition of purified mHtt（Htt-40Q）could significantly increase the enzyme digestion of purified calpain-2 on full-length CREST,which proved that mHtt could directly promote the enzyme digestion of calpian-2 on CREST.（4）CREST specific calpain-2 cleavage site blocking peptide inhibits the cytotoxicity of mHtt: after adding CREST specific calpain-2 cleavage site blocking peptide to N2 a cells transfected with Htt-20 Q or Htt-160 Q,the effects of Htt-160 Q on reducing the level of full-length CREST,promoting cell death,inhibiting the growth of N2 a cell processes,inhibiting the expression of neuronal differentiation marker MAP2,and inhibiting the phosphorylation of Rb,which can block cell cycle and promote neuronal differentiation,were significantly retarded,which further proved that CREST is a specific cleavage substrate of calpain-2,and also provides a clear scientific basis for the hypothesis that mHtt produces cytotoxicity and neuropathological changes of HD by neuronal cell cycle disorder caused by calpain degradation of CREST.（5）The carboxy terminal fragments of CREST by calpain-2 are cytotoxic: in N2 a cells transfected with full-length CREST or different fragments of CREST cleaved by calpain-2,immunofluorescence detection showed that full-length CREST was mainly dispersed in the cytoplasm and nucleus,the amino terminal fragment was mainly dispersed in the cytoplasm,while the carboxyl terminal fragment was mainly granular and located in the nuclei;Trypan blue staining and Western blot showed that the amino terminal fragments had no obvious cytotoxicity,while the carboxyl terminal fragments caused a large number of cell death and enhanced the activation of Caspase-3.These results suggest that mHtt can also cause neuropathological changes in HD by promoting calpain-2 cleavage of CREST to produce toxic carboxyl terminal fragments of CREST.The toxicity of the carboxyl terminal fragments of CREST may be related to the abnormal increase of their localization in the nuclei.Conclusion（1）mHtt can up-regulate the expression of a variety of calpains.（2）mHtt can promote the enzymatic Cleavage of CREST by calpain-2 through enhancing the interaction between calpain-2 and CREST,and then inhibit or destroy the function of CREST blocking cell cycle and promoting neuronal differentiation,resulting in cell cycle re-entry cytotoxicity of neuronal cells;The carboxy terminal fragments of CREST by of calpain-2 are toxic,and the toxicity may be related to the abnormal increase in nuclear localization of the carboxy terminal fragments.（3）CREST is a specific enzyme cleavage substrate of calpain-2.The specific enzyme cleavage site blocking peptide for CREST cleavage site can be used as a potential target for HD treatment.