Huntington disease (HD), a dominantly inherited neurodegenerative disorder caused by the expansion of a CAG-encoded polyglutamine (polyQ) repeat in huntingtin (Htt), displays a highly heterogeneous etiopathology and disease onset. Here, we show that the translation of expanded CAG repeats in mutant Htt exon 1 leads to a depletion of charged glutaminyl-transfer RNA (tRNA)(Gln-CUG) that pairs exclusively to the CAG codon. This results in translational frameshifting and the generation of various transframe-encoded species that differently modulate the conformational switch to nucleate fibrillization of the parental polyQ protein. Intriguingly, the frameshifting frequency varies strongly among different cell lines and is higher in cells with intrinsically lower concentrations of tRNA(Gln-CUG). The concentration of tRNA(Gln-CUG) also differs among different brain areas in the mouse. We propose that translational frameshifting may act as a significant disease modifier that contributes to the cell-selective neurotoxicity and disease course heterogeneity of HD on both cellular and individual levels.

In Huntington disease (HD), an expanded polyglutamine (polyQ > 37) sequence within huntingtin (htt) exon1 leads to enhanced disease risk. It has proved difficult, however, to determine whether the toxic form generated by polyQ expansion is a misfolded or avid-binding monomer, an α-helix-rich oligomer, or a β-sheet-rich amyloid fibril. Here we describe an engineered htt exon1 analog featuring a short polyQ sequence that nonetheless quickly forms amyloid fibrils and causes HD-like toxicity in rat neurons and Drosophila. Additional modifications within the polyQ segment produce htt exon1 analogs that populate only spherical oligomers and are non-toxic in cells and flies. Furthermore, in mixture with expanded-polyQ htt exon1, the latter analogs in vitro suppress amyloid formation and promote oligomer formation, and in vivo rescue neurons and flies expressing mhtt exon1 from dysfunction and death. Thus, in our experiments, while htt exon1 toxicity tracks with aggregation propensity, it does so in spite of the toxic construct's possessing polyQ tracts well below those normally considered to be disease-associated. That is, aggregation propensity proves to be a more accurate surrogate for toxicity than is polyQ repeat length itself, strongly supporting a major toxic role for htt exon1 aggregation in HD. In addition, the results suggest that the aggregates that are most toxic in these model systems are amyloid-related. These engineered analogs are novel tools for mapping properties of polyQ self-assembly intermediates and products that should similarly be useful in the analysis of other expanded polyQ diseases. Small molecules with similar amyloid inhibitory properties might be developed into effective therapeutic agents.

Triosephosphate isomerase (TPI) is a glycolytic enzyme which homodimerizes for full catalytic activity. Mutations of the TPI gene elicit a disease known as TPI Deficiency, a glycolytic enzymopathy noted for its unique severity of neurological symptoms. Evidence suggests that TPI Deficiency pathogenesis may be due to conformational changes of the protein, likely affecting dimerization and protein stability. In this report, we genetically and physically characterize a human disease-associated TPI mutation caused by an I170V substitution. Human TPII170V elicits behavioral abnormalities in Drosophila. An examination of hTPII170V enzyme kinetics revealed this substitution reduced catalytic turnover, while assessments of thermal stability demonstrated an increase in enzyme stability. The crystal structure of the homodimeric I170V mutant reveals changes in the geometry of critical residues within the catalytic pocket. Collectively these data reveal new observations of the structural and kinetic determinants of TPI deficiency pathology, providing new insights into disease pathogenesis.

Retroviral gene transfer is the method of choice for the stable introduction of genetic material into the cellular genome. However, efficient gene transfer is often limited by low transduction rates of the viral vectors. We have recently described a 12-mer peptide, termed EF-C, that forms amyloid-like peptide nanofibrils (PNF), strongly increasing viral transduction efficiencies. These nanofibrils are polycationic and bind negatively charged membranes of virions and cells, thereby overcoming charge repulsions and resulting in increased rates of virion attachment and gene transfer. EF-C PNF enhance vector transduction more efficiently than other soluble additives and offer prospects for clinical applications. However, while the transduction-enhancing activity of PNF has been well-characterized, the exact mechanism and the kinetics underlying infection enhancement as well as the cellular fate of the fibrils are hardly explored. This is partially due to the fact that current labeling techniques for PNF rely on amyloid probes that cause high background staining or lose signal intensities after cellular uptake. Here, we sought to generate EF-C PNF covalently coupled with fluorescent labels. To achieve such covalent bioconjugates, the free amino groups of the EF-C peptide were coupled to the ATTO 495 or 647N NHS ester dyes. When small amounts of the labeled peptides were mixed with a 100- to 10 000-fold excess of the native peptide, PNF formed that were morphologically indistinguishable from those derived from the unlabeled peptide. The fluorescence of the fibrils could be readily detected using fluorescence spectroscopy, microscopy, and flow cytometry. In addition, labeled and nonlabeled fibrils captured viral particles and increased retroviral transduction with similar efficacy. These covalently fluorescence-labeled PNF are valuable tools with which to elucidate the mechanism(s) underlying transduction attachment and the fate of the fibrils in cells, tissues, and animal models.