Study On Nuclear Export Mechanism Of Huntingtin And Factors Affecting Its Aggregate Formation

Huntington's Disease (HD), a neurodegenerative disease with dominant inheritance, was first reported in 1872 by George Huntington. It is characterized by motor symptoms, cognitive decline as well as neuropsychiatric abnormalities. In 1993, the gene for HD was successfully cloned and designated as IT15 gene. In normal individuals, the range of the CAG repeat is below 27; between 27 and 35, the carrier with no symptoms may transmit the disease to their children, especially in the case of paternal inheritance; people who take the CAG repeat in the range of 36 to 39 may present with an incomplete penetrance:when the repeats number is larger than 40, it is fully penetrant. Till now, there is no effective therapy. Once suffered from the disease, the patients may become disabled, or even die during the later period of 12-15 years after onset. Additionally, several family members may have the disease through their lifetime. The pathology is marked by the presence of neuronal intranuclear inclusions, as well as dystrophic neurites inclusions. The interaction between the polyglutamines (polyQ) and possibly the nuclear export deficit of huntingtin (htt) may be the culprit.With regard to study of mechanism of nuclear export of htt, the classical nuclear export signal (NES) in the carboxy-terminus and the cytoplasmic localization-related domain (CLRD) in the N-terminus of htt were both discovered. A carboxy-terminal conserved NES leucine (L)-rich sequence was detected, and further study suggested that it mediated the nuclear export of htt through a potent CRM-1/exportin export pathway. Subsequently, a CLRD, independent of the classical nuclear export pathway, was also confirmed to be located in the N-terminus of htt. Since full-length htt is cleaved at residue 513,552 or 586, and the aggregation is mainly composed of the N-terminal truncated htt fragment, suggesting that the N-terminal CLRD is more important for its cytoplasmic localization. Thus, we focus on this N-terminal CLRD in the present study. We introduced deletion and substitution to explore the essential sequence that is required for its cytoplasmic localization as well as its effect on aggregates.Abnormal CAG repeat contributes to the onset of HD, and other genetic polymorphisms in IT15 gene, like the CCG polymorphism in the N-terminus and the?2642 glutamic acid polymorphism in the C-terminus, have also been reported to be modifiers. To study the correlation between the CCG polymorphism and HD, we analyzed the CCG repeats in mainland Chinese HD families and its effect on the aggregate. Subsequently, we further investigated the effects of the proline-rich domain on the sub-cellular distribution of the protein and its aggregate formation.The composition of aggregate is complex, consisting of p53?mdm-2?HSP70?TFIID?actin?neurofilament?syntaxin 1A and nuclear pore complex as revealed by previous studies. Fully exploration of the aggregates' components may help us understand the formation process and the associated mechanism. Therefore, the co-localization analysis of htt-associated proteins and aggregates were performed preliminarily in the present study as well.Our previous work includes:1 We have collected two large HD pedigrees. On the basis of acquisition of their clinical and neuroimaging features, we further finished the IT15 gene mutation analysis in all the remaining family members. The result showed that in pedigree 1, the CAG repeat number of 9 of the 18 family members (5 patients and 4 asymptomatic subjects) reached 40; in pedigree 2, the CAG repeat number of 5 of the 24 family members (3 patients and 2 asymptomatic subjects) was more than 50;2 We constructed the plasmids carrying the wide-type and mutant-type of truncated htt fragment (pEGFP-Cl-19Q and pEGFP-Cl-70Q), both of which were transfected into non-neuronal Hela cells and neuronal SH-SY5Y cells.3 We found that sodium butyrate protected SH-SY5Y cells against death induced by mutant htt fragment. However, it did not decrease mutant htt aggregates formation, suggesting there is no essential association between htt aggregates and neuron death.4 Our previous experiment revealed that the nuclear export function of N-terminal htt fagment was eliminated in the absense of the first 5 amino acids, while still preserved in the deletion of the first 3 amino acids, indicating that Htt4-17 might be involved in the nuclear export process.Part 1 Essential sequence of the N-terminal CLRD of huntingtin and its effect on aggregatesObjectives:To confirm the key sequence of the N-terminal CLRD of htt that is required for its cytoplasmic localization; to determine which cellular apparatus the sequence directed to, and its contribution to the pathology.Methods:1 Plasmids of Htt1-17-NLS-GFP?Htt4-17-NLS-GFP?Htt5-17-NLS-GFP. Htt4-16-NLS-GFP?Htt(L4M.1-17)-NLS-GFP?Htt(M8L.1-17)-NLS-GFP?Htt(L4R.1-17)-NLS-GFP?Htt(L7R.1-17)-NLS-GFP?HttEx1P(20Q)-GFP? HttEx1p(L4R,20Q)-GFP?HttEx1P(59Q)-GFP and HttEx1P(L4R,59Q)-GFP were all constructed by recombinant DNA technology. To confirm the key residue that is required for its cytoplasmic localization, we observed the protein's intracellular distribution after transient transfection.2 Immunofluorescence was used to detect the expression and subcellular distribution of eukaryotic expression plasmids. The mitochondria, ER and Golgi were all stained by their corresponding markers.3 The effect of this sequence on the aggregate was evaluated by immunofluorescence and immunoblotting analysis after site-specific mutagenesis of both wide-type and mutant-type plasmids.Results:1 we found that the fused protein of the first 17 amino acids?NLS and GFP distributed in the cytoplasm, in contrary to either NLS-GFP or GFP, suggesting this sequence has a role of cytoplasmic localization. Deletion of the first 3 amino acids did not affect its distribution, which indicated they were not the key residues. The subcellular distribution changed when it was mutated to include amino acids of different charges (L4R or L7R), or deletion of one more amino acid from either the N-terminus or the C-terminus of Htt4-17, suggesting a structural requirement of the first 17 amino acids for the cytoplasmic localization of htt.2 The fused protein co-localized with mitochondria, rather than ER or golgi.3 L4R mutation of truncated htt fragment resulted in altered intracellular distribution and a reduction of total SDS-insoluble htt aggregates and increased nuclear aggregates.Conclusions:1 We demonstrated that the essential sequence for htt cytoplasmic localization was Htt4-17, and deletion of the first 3 amino acids did not affect the process.2 We found that this CLRD co-localized with mitochondria. 3 We prove that the absence of the first 17 amino acids resulted in a reduction of total htt aggregates and increased nuclear aggregates, further supporting its effect on aggregate formation.Part 2 CCG repeats among mainland Chinese HD families and effects of the CCG polymorphism as well as the proline-rich region on intracellular distribution of the protein and aggregatesObjectives:To improve the work of genetic family data collection, explore the CCG repeats among mainland Chinese HD families and analyze its correlation with HD; to determine the effect of the proline-rich region on intracellular distribution of the N-terminal htt fragment and its aggregates.Methods:1 Genetic family collection work includes:a. acquisition of clinical data of family members; b, phenolic alcohol/chloroform method was carried out to isolate genomic DNA from peripheral blood; c, cultured colonies of peripheral blood lymphocyte from HD family members were conducted.2 The correlation analysis was conducted between genotype (CCG polymorphisms) and phenotype (the clinical data, including sex ratio, age of onset, disease duration, family history and symptoms).3 Plasmids of exon 1 of IT15 gene with the same CAG repeat and different CCG repeat were constructed, and effects of the two polymorphisms on the intracellular distribution and quantity of the aggregates were observed after transient trasfection by both immunostaining and immunoblotting analysis.4 To determine the requirement of the CCG repeat number for htt cytoplasmic distribution, we constructed plasmids with the same CAG repeat number and different CCG repeat number (5P?7P and 9P) and observed their subcellular distributions after transient transfection.5 Plasmids with the same CAG repeat number and differently-truncated proline-rich fragment were constructed, and immunostaining and immunoblotting analysis were used to detect their effects on its intracellular distribution and aggregate formation.Results:1 53 HD families, including 60 patients, were gathered; 8 cultured colonies of peripheral blood lymphocyte from HD family members were successfully constructed. The clinical data, DNA samples as well as lymphocyte colonies were all conserved well.2 DNA sequencing confirmed the successful construction of all the above plasmids.3 In our 53 HD families studied,54.72% had 10-repeat alleles, and the left 45.28% had 7-repeat alleles; 56 normal controls (112 unrelated normal chromosomes) were also examined. The percentage of CCG(IO) and CCG(7) is 46.43% and 35.71% respectively, while CCG(9)?CCG(8) and CCG(6) is 10.71%?1.79%and 5.36%. In terms of clinical data, there was no significant difference between these two groups.4 Two plasmids of Exonl-46Q-10P-GFP and Exonl-46Q-7P-GFP were constructed. Similar intracellular distribution and aggregate quantity were observed after their expression.5 We also found that the protein presented a cytoplasmic distribution only with 7 CCG repeats, rather than with 5 CCG repeats, indicating that the CCG repeat number affected its intracellular distribution.6 Compared with Exonl-20Q/59Q-GFP, the distribution of 1-17-20Q/59Q-GFP was more in the nucleus, which could be reversed by adding of the first proline-rich region, further supporting the role of this region in its intracellular localization. Similarly, compared with Exonl-59Q-10P-GFP, aggregates formed by 1-17-59Q-GFP were smaller and more scattered, which could be reversed by adding the first proline-rich region. Western blot shows that the ratio of aggregate to soluble protein is significantly lowest in cells transfected by 1-17-59Q-GFP.Conclusion:1 We find that the 7-CCG repeat and the 10-CCG repeat are the most frequent (45.28% and 54.72%, respectively) types of mutant alleles in mainland Chinese HD families;2 We find that the two polymorphisms did not differ from the clinical information to the cell model, which suggests that this polymorphism is not associated with HD.3 In addition to confirmation of the role of the proline-rich region in the protein's distribution, our study suggests the key role that the first proline-rich region plays for the first time; we also point out the requirement of 7 CCG repeats for its cytoplasmic distribution.4 In addition to confirming that the aggregates become smaller and more scattered after deletion of the proline-rich region, we also find that it can be reversed by adding the first proline-rich region Part 3 Exploration of the component of the aggregateObjectives:To explore the component of the aggregate, and to provide some preliminary data for future study of HD.Methods:Laser scanning confocal microscope was used to observe the relationship between the aggregate and ubiquitin?vimentin?caspase-3?caspase-9?HSP-70.Results:Ubiquitin?vimentin?caspase-3?caspase-9 and HSP-70 were all co-localized with the aggregates, suggesting that the aggregate composition is complex.Conclusions:1) We confirmed that ubiquitin?vimentin and HSP-70 were all included in the aggregate.2) For the first time we point out that caspase-3 and caspase-9 are also the composition of aggregate.