Huntington's Pathogenicity regulated by Cellular Translation Ankita Deo1, Mrunalini Shinde1, Ruta Chitale1, Meghal Desai2, Vighnesh Ghatpande2, Maitheli Sarkar2, Amitabha Majumadar2, Tania Bose1 1 Institute of Bioinformatics and Biotechnology, Savitribai Phule Pune University, Pune 2 National Centre for Cell Science, Savitribai Phule Pune University, Pune
Orb2 sequesters with Huntington aggregates
Abstract Background: Huntington’s disease (Htt) is an effect of an autosomal dominant mutation on short arm of chromosome four affecting 1 in every 20,000 individuals. 40,000-70,000 people have been affected by this condition in India. Rationale: Studies have been done where chaperones and certain drug molecules are used to reduce the protein aggregation which is hallmark of the disease but exact mode of action of the mutation is unknown. We have studied the mechanism where Huntington’s disease is seen affecting protein translation and have used a translational regulator to minimize the effects of mutation. Strategy: Huntington’s mutation was mimicked in drosophila cells and S.cerevisiae by introducing extended repeats of CAG (PolyQ tract) in them. Puromycin incorporation experiment was used to study the crosstalk between the Htt aggregates and translational machinery. A translational regulator (Orb2) was seen sequestering with mutant aggregates in FRAP experiments and was seen to improve translational deficit of mutant in Polysome profiling. Results: Orb2 was seen to sequester with Huntington’s aggregates. Pan-neuronal expression of mutation (Htt 138Q) was seen to be lethal and could be rescued by isoforms of Orb2 whereas Orb2 knockdown decreased the recovery rate. On quantitation of Htt aggregates, there was no difference in size of Htt138Q and Orb2 rescued cells but Polysome profiling showed that Orb2 co-expression recues deficit in Polysome:80S and improves translatory status of the mutant cell. Conclusion: Huntington aggregates sequester translational machinery of the cell, thereby affecting translation. This effect can be minimized by co-expression of Orb2, a translational regulator.
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Huntington’s Pathogenicity CAG
CAG
CAG
CAG
Mutation on short arm of chromosome four
CAG n
Expansion of PolyQ tract Symptomized by involuntary movements, psychological abnormalities,cognitive dysfunction and neuronal loss
Poly Glutamine
(CAG)n Translational Deficit
HD allele ≥40 40 HD allele with reduced penetrance (36-39)
36 27
F
E
Mutable normal allele (27-35)
Htt aggregates Sequestered proteins
Fig3.A.Orb2 is seen co-localizing with RFP tagged Htt103Q in S.cerevisiae cells observed under confocal microscope. B.Left panels show Drosophila optic lobes expressing GFP tagged Orb2A, Orb2B and RFP tagged HttQ138 with DAPI in blue color. Right panels show optic lobes coexpressing Orb2A and Orb2B with HttQ138. Upon coexpression, Orb2A and Orb2B are sequestered in HttQ138 aggregates. C.Quantitation of relative Orb2 levels shows increased Orb2B in HttQ138 expressing Drosophila heads. D.Immunoprecipitation from Elav; HttQ15 and Elav; HttQ138 Drosophila heads with anti Orb2 antibody and probed with an antiHtt antibody shows Orb2 pulls down HttQ138 but not HttQ15. E,F. Fluorescence recovery after photobleaching (FRAP) experiment data shows signifcantly decreased recovery for sequestered Orb2A and Orb2B in the presence of HttQ138 in comparison to only Orb2A and Orb2B expressing cells.
Deficit repaired
Normal allele ≤26
Orb2 rescues translational deficit of Huntington mutants
Orb2
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Yeast and S2 cell lines as a model for studying translation in Huntington’s disease
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A 25Q
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103Q
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Fig.4. A.Eclosion experiments show that coexpression of Orb2A and Orb2B can rescue the lethality associated with pan-neuronal expression of HttQ138 using Elav Gal4. Eclosion experiments performed with a lower expressing Elav Gal4 line show that in the presence of Orb2RNAi coexpression there is decreased survival of HttQ138 expressing animals. B.Quantitation of aggregate numbers from HttQ138 alone and HttQ138 with Orb2A or Orb2B from motor neurons do not show any signifcant diference. C.Representative SDD-AGE immunoblot showing no signifcant diference between the Htt oligomers in HttQ138 and HttQ138 with Orb2A and Orb2B. D.Quantitation of Polysome/80S ratio from HttQ138 and HttQ138+Orb2A GFP and HttQ138+ Orb2B GFP expressing cells shows that coexpression of Orb2A and Orb2B rescues the reduced polysome/80S ratio associated with HttQ138 cells. E.Quantitation of Puromycin incorporation from HttQ138 and HttQ138+ Orb2A GFP and HttQ138+Orb2B GFP expressing cells shows signifcantly reduced Puromycin incorporation in HttQ138 cells and rescue with coexpression of Orb2A and Orb2B
Ribosomal proteins sequester with Huntington aggregates 4
A Fig.1 A. Spot dilution test of normal and mutant S.crevisiae cells where the mutant is with an expanded polyQ tract. Mutant 103Q is seen to be growing slowly than wildtype 25Q as the effect of mutation. B. Polysome profiles of S2 cells expressing Htt 15Q (wildtype) and Htt 138Q (mutant) tagged with RFP (inset). Quantitation of Polysome/80S ratio data. C.Polysome profles of Yeast expressing HttQ25 and HttQ103. Inset show images of cells expressing GFP tagged HttQ25 and HttQ103. Quantitation of Polysome/80S ratio WT scc2-D730V from HttQ25 and HttQ103 expressing Yeast.
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RPL-25
Q25
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C eif2α-P eif2α
Puromycin Incorporation decreased in the mutants A
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RPL-25
Q103
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Fig.5. A. RPL-25, a protein associated with ribosome assembly, was also seen to be sequestered with Huntington’s aggregates in S. cerevisiae cells just like Orb2. B.Polysome profiling of yeast cells show translational deficit. C.Eif2α phosphorylation was checked in Drosophila strains Q15 and Q138, which showed increased phosphorylation in the mutant (Q138) indicating translation initiation defect.
Conclusion: Huntington’s disease causes protein aggregates that sequester RNA bindng proteins, ribosome
Fig2.A. Puromycin incorporation assay was performed using anti Puromycin antibody. Western blot shows reduced Puromycin incorporation in HttQ138 cells in comparison to HttQ15 cells. The right panel is the Ponceau-S stained membrane for the same blot. B.Quantitation of Puromycin incorporation from HttQ15 and HttQ138 cells shows signifcantly reduced Puromycin incorporation in HttQ138 cells. C.O-Propargyl-Puromycin(OPP) incorporation assay shows incorporation of OPP in HttQ138 aggregates
associated proteins and translational regulators. Polysome profiling of these cells show that translation of the cells is affected. Eif2α phosphorylation increases in mutant indicating a translational initiation defect. Co-expressing these cells with translational regulator like Orb2 rescues their translational deficit and reduces the lethality caused by the mutation thereby improving translatory status of the cells.