Allosteric Modulation of Pathological Ataxin-3 Aggregation: A Path to Spinocerebellar Ataxia Type-3 Therapies

Abstract

Spinocerebellar ataxia type 3 (SCA3) is a rare inherited neurodegenerative disease caused by the expansion of a polyglutamine repeat in the protease ataxin-3 (Atx3). Despite extensive knowledge of the downstream pathophysiology, no disease-modifying therapies are currently available to halt disease progression. The accumulation of protein inclusions enriched in the polyQ-expanded Atx3 in neurons suggests that inhibiting its self-assembly may yield targeted therapeutic approaches. Here it is shown that a supramolecular tweezer, CLR01, binds to a lysine residue on a positively charged surface patch of the Atx3 catalytic Josephin domain. At this site, the binding of CLR01 decreases the conformational fluctuations of the distal flexible hairpin. This results in reduced exposure of the nearby aggregation-prone region, which overlaps with the substrate ubiquitin binding site and primes Atx3 self-assembly, ultimately delaying Atx3 amyloid fibril formation and reducing the secondary nucleation rate, a process linked to fibril proliferation and toxicity. These effects translate into the reversal of synapse loss in a SCA3 cultured cortical neuron model, an improved locomotor function in a C. elegans SCA3 model, and a delay in disease onset, accompanied by reduced severity of motor symptoms in a SCA3 mouse model. This study provides critical insights into Atx3 self-assembly, revealing a novel allosteric site for designing CLR01-inspired therapies targeting pathological aggregation pathways while sparing essential functional sites. These findings emphasize that targeting allosteric sites in amyloid-forming proteins may offer unique opportunities to develop safe therapeutic strategies for various protein misfolding disorders.

Keywords: Amyloid; Molecular therapies; Polyglutamine; Pre-clinical models; Protein dynamics.

Figure 1 –. CLR01 interacts with Atx3 and induces conformational changes in the JD.

a) Chemical structure of the molecular tweezer CLR01 and the control molecule CLR03. b) ESI-MS spectra of wild-type Atx3 (Atx3 13Q) and CLR01 at a 1:1 molar ratio. The inset shows the deconvoluted spectra and highlights the two CLR01 binding events. c) Cartoon representation of three states of JD: Experimental NMR models of the JD in open (PDB ID: 1YZB,[33] yellow) and closed (PDB ID: 2AGA,[34] orange) conformations. A representative structure (light pink) of the “half-open” conformation of the JD is shown (most populated cluster from the GaMD simulations in complex with CLR01). d) Amino acid sequence of JD with residues undergoing chemical shifts in the NMR HSQC spectra upon CLR01 binding in red. Secondary structure elements in the JD are shown below the amino acid sequence with helix α1 in green, the aggregation-prone segment (α4-β1) in bold and orange, and helix α6 in teal. e) Cartoon representation of the JD open conformation with the residues affected by CSP upon addition of CLR01 shown as red spheres. K128, a key residue in the interaction between CLR01 and JD, is highlighted as pink spheres. Other regions are colored as in panel d). f) Cartoon representation of the third most populated cluster from the GaMD simulations of JD (open conformation) with a single CLR01 molecule (magenta sticks) bound at K128 (pink spheres). Residues displaying a chemical shift in the HSQC spectrum are highlighted as red spheres; CLR01 is represented as dark pink sticks; R182 and other positively charged residues, corresponding to predicted CLR01 binding sites according to GaMD simulations and Gibbs binding energy estimations, are shown as sticks. This structure highlights a state where the hairpin (yellow surface) moves toward the aggregation-prone region (orange).

Figure 2 -. CLR01 binding modulates the Atx3 aggregation pathway, delaying amyloid fibril assembly and decreasing the secondary nucleation rate.

ThT measurement of 5 μM Atx3 13Q (a) and Atx3 77Q (b) amyloid self-assembly kinetics in the absence or presence of CLR01 or CLR03. Error bars correspond to the standard deviation of three replicates. c) Time-dependent DLS intensity distributions during the aggregation of 5 μM wild-type Atx3 alone or in the presence of 25 μM CLR01 or CLR03. Different colors show different time points. Vertical lines: scattering intensities predicted by a nucleation-and-growth model for monomers (thinner lines) and amyloid fibrils (thicker lines) at the end of 48 h incubation (refer to Supporting Information Figure S7 for other time points); d) Same as c) for Atx3 77Q. e) Fitted nucleation-and-growth rate constants (Table 1).

Figure 3 –. CLR01 reduces the assembly of mature fibrils of mutant Atx3 and dissociates pre-formed protofibrils and mature fibrils.

a) Morphological analysis of 5 μM Atx3 13Q or Atx3 77Q aggregates in the absence or presence of 50 μM CLR01 or CLR03 at the endpoint of the aggregation assay, monitored by negative staining TEM. Scale bars correspond to 100 nm; orange rectangles denote Atx3 curvilinear protofibrils; red rectangles highlight small protofibrils formed in the presence of CLR01, red triangles point to Atx3 spherical oligomers; white arrowheads mark clusters of spherical aggregates; and white open arrows point to mature Atx3 Q77 fibrils. b) Detection of mature SDS-resistant fibrils at the endpoint of 5 μM Atx3 77Q aggregation in the presence of 5- or 10-fold molar excess of CLR01 or CLR03 by filter retardation followed by immunoblot using anti-Atx3 1H9 antibody. A, B, and C are sample replicates. c) Morphological analysis of 5 μM Atx3 13Q or Atx3 77Q protofibrils at t=0h and after three days of incubation at 37 °C in the absence (Control) or presence of 200 μM CLR01 or CLR0 by negative-staining TEM. Scale bars correspond to 100 nm. d) Morphological analysis of 5 μM 77Q mature SDS-resistant fibrils at T0h and after ten days of incubation at 37 °C in the absence (Control) and presence of 200 μM CLR01 by negative-staining TEM. Scale bars correspond to 200 nm.

Figure 4 -. CLR01 reverts the loss of glutamatergic synapses in cortical neurons caused by pathological Atx3.

Rat cortical neurons were transfected with plasmids encoding eGFP, wild-type eGFP-Atx3 28Q, or mutant eGFP-Atx3 84Q. a) Neurons were immunolabeled for MAP2, PSD95, and vGLUT1. Excitatory synapses were detected as PSD95 puncta that colocalized with vGLUT1; scale bar = 10 μm. b) Integrated fluorescence intensity of PSD95 puncta colocalized with vGLUT1 (n= 33-36 neurons per condition in 3 independent experiments). Statistical analyses were performed using Kruskal–Wallis and Dunn’s post hoc test (Supporting Information Table S2). c) Neurons incubated with 10 μM CLR03 or CLR01 were immunolabelled for MAP2, PSD95 and VGluT1 clusters; scale bar = 10 μm. d) Integrated fluorescence intensity of PSD95 puncta colocalized with VGluT1 (n=35-41 neurons per condition in each of 3 independent experiments. Statistical analyses were performed using Kruskal–Wallis and Dunn’s post hoc tests (Supporting Information Table S3). In panels c) and d), boxes show the 25th and 75th percentiles, whiskers range from the minimum to the maximum values, and the horizontal line shows the median value. Mean is represented by “+”. e) GFP-Atx3 accumulates in the cell body of a fraction of transfected cortical cultures treated with CLR03 or CLR01; scale bars= 300 μm, 15 μm. f) Percentage of cells with GFP-Atx3 aggregates (n=3 independent experiments, 31-35 neurons per condition). Statistical analyses were performed using Two-way ANOVA and Sidak's multiple comparisons post hoc tests (Supporting Information Table S4). p-values are indicated in the graphs shown in panels b), d), and f). Data are represented as mean ± SEM.

Figure 5 -. CLR01 treatment alleviates motor deficits in a C. elegans model of SCA3 even when treatment is initiated at a post-symptomatic disease phase.

a) CLR01 treatment improved motor dysfunction of mutant Atx3 animals. AT3q130 animals were treated with different concentrations of CLR01 for four days, and the percentage of locomotion-defective animals was determined. CLR01 significantly impacted motor phenotype between 0.0001 and 1 μM; the maximum efficiency (%) was observed at 0.1 μM at which 67% phenotypic rescue was achieved compared to the control strain (Supporting Information Table S6). b) Treatment with 0.1 μM CLR01 for four days did not impact motor phenotype in the wild-type strain (N2). c) Post-symptomatic treatment with CLR01 improves the animals' motor defects after 2 or 4 days of treatment (days 6 and 8 after hatching). (a-c) Data are represented as mean ± SD of 4 independent experiments, with ~50 animals per condition/per assay (total number of animals of ~250). A one-way ANOVA test was applied, followed by a bilateral Dunnet test for post hoc comparisons (a and c). In the comparison between the two groups (b, and c- before treatment), independent-samples t-test was used (Supporting Information Table S7 and S8). d-g) Impact of early life chronic CLR01 treatment on mutant human Atx3 aggregation pattern, labeled with an Atx3-specific antibody in the C. elegans model of SCA3 throughout animals’ development and adulthood. Pictures of the animal's heads were obtained by Confocal Microscopy (Olympus FV1000) and analyzed using the MeVisLab software [48]. The graphs show a pool of 3 independent assays, with at least four animals per condition in each trial. P-values were calculated using independent-samples t-test (Supplementary Information Table S8). Scale bars = 20 μm.

Figure 6 -. CLR01 chronic administration improves motor function and neuropathology of SCA3 mice.

a) the latency to cross a swimming tank, representing both the strength and the movement coordination ability of the animals, is higher in vehicle-treated SCA3 mice. The severity of this symptom improves over time with CLR01 treatment, which has a delayed onset and is effective up to 16 weeks of age.; b) CLR01 improves SCA3 mice balance, decreasing their latency to cross a 12 mm-squared beam. c) The gait quality of SCA3 animals is strikingly improved by CLR01 treatment, delaying the onset of ataxic gait by eight weeks. d) Representative images of Cresyl Violet staining in the cervical spinal cord of healthy and pyknotic cells. e) Vehicle-treated SCA3 mice showed a substantial increase in the number of pyknotic cells, which was rescued by daily CLR01 administration for 18 weeks. f) Representative pictures of the motor-neuron cell bodies (black arrows) in the ventral horn of the cervical spinal cord. g) CLR01 treatment increases the number of motor neurons in the cervical spinal cord. Sample size and statistical details are provided in Supporting Information, Table S9. The scale bar for lower magnification pictures represents 200 μm, and for higher magnification, 100 μm. P-values are shown in the graph. Black lettering represents the comparisons between WT-vehicle and SCA3-vehicle. Red lettering represents the comparisons between SCA3-vehicle and SCA3-CLR01.

Figure 7 –. Schematic representation of the dual action of CLR01 on the delay of Atx3 self-association (1) and fibril disruption (2).

Binding of CLR01 to K128 (colored dark blue) within a positively charged surface patch opposite to the aggregation-prone region (orange) triggers a conformational change in the helical hairpin (yellow) of the Josephin domain. The CLR01-induced conformational change reshapes the Atx3 aggregation pathway and reduces secondary nucleation rates. This modulation of the self-assembly pathway correlates with the reversion of synapse dysfunction in SCA3 cell models and a delay in the onset of motor impairment in SCA3 animal models. The spherical oligomers observed upon incubation with CLR01 may be on-pathway to protofibril assembly or result from fibril dissociation processes. Created in BioRender (https://BioRender.com/e62f696).