RNA Toxicity and Perturbation of rRNA Processing in Spinocerebellar Ataxia Type 2.

BACKGROUND: Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease caused by expansion of a CAG repeat in Ataxin-2 (ATXN2) gene. The mutant ATXN2 protein with a polyglutamine tract is known to be toxic and contributes to the SCA2 pathogenesis. OBJECTIVE: Here, we tested the hypothesis that the mutant ATXN2 transcript with an expanded CAG repeat (expATXN2) is also toxic and contributes to SCA2 pathogenesis. METHODS: The toxic effect of expATXN2 transcripts on SK-N-MC neuroblastoma cells and primary mouse cortical neurons was evaluated by caspase 3/7 activity and nuclear condensation assay, respectively. RNA immunoprecipitation assay was performed to identify RNA binding proteins (RBPs) that bind to expATXN2 RNA. Quantitative PCR was used to examine if ribosomal RNA (rRNA) processing is disrupted in SCA2 and Huntington's disease (HD) human brain tissue. RESULTS: expATXN2 RNA induces neuronal cell death, and aberrantly interacts with RBPs involved in RNA metabolism. One of the RBPs, transducin ß-like protein 3 (TBL3), involved in rRNA processing, binds to both expATXN2 and expanded huntingtin (expHTT) RNA in vitro. rRNA processing is disrupted in both SCA2 and HD human brain tissue. CONCLUSION: These findings provide the first evidence of a contributory role of expATXN2 transcripts in SCA2 pathogenesis, and further support the role of expHTT transcripts in HD pathogenesis. The disruption of rRNA processing, mediated by aberrant interaction of RBPs with expATXN2 and expHTT transcripts, suggest a point of convergence in the pathogeneses of repeat expansion diseases with potential therapeutic implications. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Partially Unfolded Forms of the Prion Protein Populated under Misfolding-promoting Conditions: CHARACTERIZATION BY HYDROGEN EXCHANGE MASS SPECTROMETRY AND NMR.

The susceptibility of the cellular prion protein (PrP(C)) to convert to an alternative misfolded conformation (PrP(Sc)), which is the key event in the pathogenesis of prion diseases, is indicative of a conformationally flexible native (N) state. In the present study, hydrogen-deuterium exchange (HDX) in conjunction with mass spectrometry and nuclear magnetic resonance spectroscopy were used for the structural and energetic characterization of the N state of the full-length mouse prion protein, moPrP(23-231), under conditions that favor misfolding. The kinetics of HDX of 34 backbone amide hydrogens in the N state were determined at pH 4. In contrast to the results of previous HDX studies on the human and Syrian hamster prion proteins at a higher pH, various segments of moPrP were found to undergo different extents of subglobal unfolding events at pH 4, a pH at which the protein is known to be primed to misfold to a ß-rich conformation. No residual structure around the disulfide bond was observed for the unfolded state at pH 4. The N state of the prion protein was observed to be at equilibrium with at least two partially unfolded forms (PUFs). These PUFs, which are accessed by stochastic fluctuations of the N state, have altered surface area exposure relative to the N state. One of these PUFs resembles a conformation previously implicated to be an initial intermediate in the conversion of monomeric protein into misfolded oligomer at pH 4.

Thermodynamic characterization of the unfolding of the prion protein.

The prion protein appears to be unusually susceptible to conformational change, and unlike nearly all other proteins, it can easily be made to convert to alternative misfolded conformations. To understand the basis of this structural plasticity, a detailed thermodynamic characterization of two variants of the mouse prion protein (moPrP), the full-length moPrP (23-231) and the structured C-terminal domain, moPrP (121-231), has been carried out. All thermodynamic parameters governing unfolding, including the changes in enthalpy, entropy, free energy, and heat capacity, were found to be identical for the two protein variants. The N-terminal domain remains unstructured and does not interact with the C-terminal domain in the full-length protein at pH 4. Moreover, the enthalpy and entropy of unfolding of moPrP (121-231) are similar in magnitude to values reported for other proteins of similar size. However, the protein has an unusually high native-state heat capacity, and consequently, the change in heat capacity upon unfolding is much lower than that expected for a protein of similar size. It appears, therefore, that the native state of the prion protein undergoes substantial fluctuations in enthalpy and hence, in structure.

Stick-slip unfolding favors self-association of expanded HTT mRNA.

In Huntington's Disease (HD) and related disorders, expansion of CAG trinucleotide repeats produces a toxic gain of function in affected neurons. Expanded huntingtin (expHTT) mRNA forms aggregates that sequester essential RNA binding proteins, dysregulating mRNA processing and translation. The physical basis of RNA aggregation has been difficult to disentangle owing to the heterogeneous structure of the CAG repeats. Here, we probe the folding and unfolding pathways of expHTT mRNA using single-molecule force spectroscopy. Whereas normal HTT mRNAs unfold reversibly and cooperatively, expHTT mRNAs with 20 or 40 CAG repeats slip and unravel non-cooperatively at low tension. Slippage of CAG base pairs is punctuated by concerted rearrangement of adjacent CCG trinucleotides, trapping partially folded structures that readily base pair with another RNA strand. We suggest that the conformational entropy of the CAG repeats, combined with stable CCG base pairs, creates a stick-slip behavior that explains the aggregation propensity of expHTT mRNA.

Ruggedness in the Free Energy Landscape Dictates Misfolding of the Prion Protein.

Experimental determination of the key features of the free energy landscapes of proteins, which dictate their adeptness to fold correctly, or propensity to misfold and aggregate and which are modulated upon a change from physiological to aggregation-prone conditions, is a difficult challenge. In this study, sub-millisecond kinetic measurements of the folding and unfolding of the mouse prion protein reveal how the free energy landscape becomes more complex upon a shift from physiological (pH 7) to aggregation-prone (pH 4) conditions. Folding and unfolding utilize the same single pathway at pH 7, but at pH 4, folding occurs on a pathway distinct from the unfolding pathway. Moreover, the kinetics of both folding and unfolding at pH 4 depend not only on the final conditions but also on the conditions under which the processes are initiated. Unfolding can be made to switch to occur on the folding pathway by varying the initial conditions. Folding and unfolding pathways appear to occupy different regions of the free energy landscape, which are separated by large free energy barriers that change with a change in the initial conditions. These barriers direct unfolding of the native protein to proceed via an aggregation-prone intermediate previously identified to initiate the misfolding of the mouse prion protein at low pH, thus identifying a plausible mechanism by which the ruggedness of the free energy landscape of a protein may modulate its aggregation propensity.

Identification and Structural Characterization of the Precursor Conformation of the Prion Protein which Directly Initiates Misfolding and Oligomerization.

To identify and structurally characterize the precursor conformation of the prion protein (PrP), from which misfolding and aggregation directly commence, has been a long-standing goal. Misfolding converts the α-helical, non-pathogenic functional form of PrP to pathogenic, ß-structured oligomeric and amyloidogenic forms, which are the cause of prion diseases. Susceptibility to sporadic prion disease correlates well with the propensity of PrP to misfold to cytotoxic, proteinase K resistant oligomeric conformations at low pH. In this study, mutagenesis at the hydrophobic core of the mouse PrP has been shown to stabilize a monomeric unfolding intermediate (I), which is populated significantly at equilibrium at low pH. Importantly, the rate of formation of ß-structured oligomers at low pH is found to correlate well with the extent to which this intermediate is populated. The misfolding process is limited by the dimerization of I, indicating that I is the monomeric precursor conformation that directly initiates misfolding. Structural and thermodynamic characterization by native-state hydrogen-deuterium exchange mass spectrometry studies indicate that the precursor conformation is a partially unfolded form of PrP that forms under misfolding-prone solvent conditions.