Inhibition of Aggregation of Mutant Huntingtin by Nucleic Acid Aptamers In Vitro and in a Yeast Model of Huntington's Disease.

Elongated polyglutamine stretch in mutant huntingtin (mhtt) correlates well with the pathology of Huntington's disease (HD). Inhibition of aggregation of mhtt is a promising strategy to arrest disease progression. In this work, specific, high-affinity RNA aptamers were selected against monomeric mhtt (51Q-htt). Some of them inhibited its aggregation in vitro by stabilizing the monomer. They also recognized 103Q-htt but not 20Q-htt (nonpathogenic length). Inhibition of aggregation corresponded with reduced leakage of a fluorescent probe from liposomes and diminished oxidative stress in RBCs. The presence of aptamers was able to rescue the sequestration of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by aggregated mhtt. Some of the aptamers were able to enhance the partitioning of mhtt in the soluble fraction in a yeast model of HD. They were also able to rescue endocytotic defect due to aggregation of mhtt. The beneficial effect of a combination of aptamers was enhanced with improvement in cell survival. Since HD is a monogenic autosomal dominant disorder, aptamers may be developed as a viable strategy to slow down the progress of the disease. Since they are nonimmunogenic and nontoxic, aptamers may emerge as strong candidates to reduce protein-protein interaction and hence protein aggregation in protein misfolding disorders in general.

RNA Aptamers Rescue Mitochondrial Dysfunction in a Yeast Model of Huntington's Disease.

Huntington's disease (HD) is associated with the misfolding and aggregation of mutant huntingtin harboring an elongated polyglutamine stretch at its N terminus. A distinguishing pathological hallmark of HD is mitochondrial dysfunction. Any strategy that can restore the integrity of the mitochondrial environment should have beneficial consequences for the disease. Specific RNA aptamers were selected that were able to inhibit aggregation of elongated polyglutamine stretch containing mutant huntingtin fragment (103Q-htt). They were successful in reducing the calcium overload, which leads to mitochondrial membrane depolarization in case of HD. In one case, the level of Ca2+ was restored to the level of cells not expressing 103Q-htt, suggesting complete recovery. The presence of aptamers was able to increase mitochondrial mass in cells expressing 103Q-htt, along with rescuing loss of mitochondrial genome. The oxidative damage to the proteome was prevented, which led to increased viability of cells, as monitored by flow cytometry. Thus, the presence of aptamers was able to inhibit aggregation of mutant huntingtin fragment and restore mitochondrial dysfunction in the HD cell model, confirming the advantage of the strategy in a disease-relevant parameter.

Specific detection of tetanus toxoid using an aptamer-based matrix.

Batch-to-batch variation of therapeutic proteins produced by biological means requires rigorous monitoring at all stages of the production process. A large number of animals are employed for risk assessment of biologicals, which has low ethical and economic acceptability. Research is now focussed on the validation of in vitro and ex vivo tests to replace live challenges. Among in vitro methods, enzyme-linked immunosorbent assay (ELISA) is considered to be the gold standard for estimation of integrity of tetanus toxoid. ELISA utilizes antibodies for detection, which, because of their biological origin and limited modifiability, may have low stability and result in irreproducibility. We have developed a method using highly specific and selective RNA aptamers for detection of tetanus toxoid. Using displacement assay, we first identified aptamers which bind to different aptatopes on the surface of the toxoid. Pairs of these aptamers were employed as capture-detection ligands in a sandwich-ALISA (aptamer-linked immobilized sorbent assay) format. The binding efficiency was confirmed by the fluorescence intensity in each microtire plate well. Using aptamers alone, detection of tetanus toxoid was possible with the same level of sensitivity as antibody. Aptamers were also used in the capture ALISA format. Adjuvanted tetanus toxoid was subjected to accelerated stress testing, including thermal, mechanical and freeze-thawing stress conditions. The loss in antigenicity of the preparation determined by ALISA in each case was found to be similar to that determined by conventional ELISA. Thus, it is possible to replace antibodies with aptamers to develop a more robust detection tool for tetanus toxoid.

Effect of pesticides on the aggregation of mutant huntingtin protein.

The classical reports on neurodegeneration concentrate on studying disruption of signalling cascades. Although it is now well recognized that misfolding and aggregation of specific proteins are associated with a majority of these diseases, their role in aggravating the symptoms is not so well understood. Huntington's disease (HD) is a neurodegenerative disorder that results from damage to complex II of mitochondria. In this work, we have studied the effect of mitochondrial complex I inhibitors, viz. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and rotenone, and complex II inhibitor, viz. 3-nitropropionic acid, on the aggregation of mutant huntingtin (mthtt) protein, whose misfolding and aggregation results in cellular abnormalities characteristic of HD. All three inhibitors were found to accelerate the aggregation of mthtt in vitro, although the amounts of aggregates formed were different in all cases. Thus, apart from their effect on mitochondrial viability, these neurotoxins are capable of interfering with the protein aggregation process and thus, hastening the onset of the disease.