Spinocerebellar ataxia type 5 (SCA5) mutations in the protein β-III-spectrin cluster to the N-terminal actin-binding domain (ABD) and the central spectrin-repeat domains (SRDs). We previously reported that a common molecular consequence of ABD-localized SCA5 mutations is increased actin binding. However, little is known about the molecular consequences of the SRD-localized mutations. It is known that the SRDs of β-spectrin proteins interact with α-spectrin to form an α/β-spectrin dimer. In addition, it is known that SRDs neighboring the β-spectrin ABD enhance actin binding. Here, we tested the impact of the SRD-localized R480W and E532_M544del mutations on the binding of β-III-spectrin to α-II-spectrin and actin. R480W is associated with a severe infantile onset form of SCA5, while E532_M544del is associated with milder symptoms that begin in adulthood. We show that both the R480W and E532_M544del mutants can bind α-II-spectrin. However, E532_M544del causes partial uncoupling of complementary SRDs in the α/β-spectrin dimer. Further, the R480W mutant forms large intracellular inclusions when coexpressed with α-II-spectrin in cells, supporting that R480W grossly disrupts the α-II/β-III-spectrin complex. Moreover, actin-binding assays show that E532_M544del, but not R480W, increases β-III-spectrin actin binding. Additionally, we demonstrate that R480W α-II/β-III-spectrin inclusions contain F-actin, accumulate the spectrin-binding protein ankyrin-R, and localize immediately adjacent to the Golgi complex. Two additional infantile onset mutations, R437W and R437Q, but not the adult onset T472M mutation, also cause formation of large α-II/β-III-spectrin inclusions. We suggest that the intracellular inclusions caused by R480W, R437W, and R437Q drive the more severe disease symptoms associated with these mutations.

Spinocerebellar ataxia type 5 (SCA5) mutations in the protein β-III-spectrin cluster to the N-terminal actin-binding domain (ABD) and the central spectrin-repeat domains (SRDs). We previously reported that a common molecular consequence of ABD-localized SCA5 mutations is increased actin binding. However, little is known about the molecular consequences of the SRD-localized mutations. It is known that the SRDs of β-spectrin proteins interact with α-spectrin to form an α/β-spectrin dimer. In addition, it is known that SRDs neighbouring the β-spectrin ABD enhance actin binding. Here, we tested the impact of the SRD-localized R480W and the E532_M544del mutations on the binding of β-III-spectrin to α-II-spectrin and actin. Using multiple experimental approaches, we show that both the R480W and E532_M544del mutants can bind α-II-spectrin. However, E532_M544del causes partial uncoupling of complementary SRDs in the α/β-spectrin dimer. Further, the R480W mutant forms large intracellular inclusions when co-expressed with α-II-spectrin in cells, supporting that R480W mutation grossly disrupts the α-II/β-III-spectrin physical complex. Moreover, actin-binding assays show that E532_M544del, but not R480W, increases β-III-spectrin actin binding. Altogether, these data support that SRD-localized mutations alter key interactions of β-III-spectrin with α-II-spectrin and actin.

Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the SPTBN2 gene encoding the cytoskeletal protein β-III-spectrin. Previously, we demonstrated that a L253P missense mutation, localizing to the β-III-spectrin actin-binding domain (ABD), causes increased actin-binding affinity. Here we investigate the molecular consequences of nine additional ABD-localized, SCA5 missense mutations: V58M, K61E, T62I, K65E, F160C, D255G, T271I, Y272H, and H278R. We show that all of the mutations, similar to L253P, are positioned at or near the interface of the two calponin homology subdomains (CH1 and CH2) comprising the ABD. Using biochemical and biophysical approaches, we demonstrate that the mutant ABD proteins can attain a well-folded state. However, thermal denaturation studies show that all nine mutations are destabilizing, suggesting a structural disruption at the CH1-CH2 interface. Importantly, all nine mutations cause increased actin binding. The mutant actin-binding affinities vary greatly, and none of the nine mutations increase actin-binding affinity as much as L253P. ABD mutations causing high-affinity actin binding, with the notable exception of L253P, appear to be associated with an early age of symptom onset. Altogether, the data indicate that increased actin-binding affinity is a shared molecular consequence of numerous SCA5 mutations, which has important therapeutic implications.

Nusinersen has been shown to improve or stabilize motor function in individuals with spinal muscular atrophy (SMA). We evaluated baseline scoliosis severity and motor function in nusinersen-treated non-ambulatory children with later-onset SMA. Post hoc analyses were conducted on 95 children initiating nusinersen treatment in the CHERISH study or SHINE long-term extension trial. Participants were categorized by baseline Cobb angle (first nusinersen dose): ≤10°, >10° to ≤20°, and >20° to <40° (no/mild/moderate scoliosis, respectively). Outcome measures included the Hammersmith Functional Motor Score-Expanded (HFMSE) and the Revised Upper Limb Module (RULM). Regression analysis determined the relationships between baseline scoliosis severity and later motor function. For children with no, mild, and moderate scoliosis, the mean increase in HFMSE from baseline to Day 930 was 6.0, 3.9, and 0.7 points, and in RULM was 6.1, 4.6, and 2.3 points. In the linear model, a 10° increase in baseline Cobb angle was significantly associated with a -1.4 (95% CI -2.6, -0.2) point decrease in HFMSE (p = 0.02) and a -1.2 (95% CI -2.1, -0.4) point decrease in RULM (p = 0.006) at Day 930. Treatment with nusinersen was associated with improvements/stabilization in motor function in all groups, with greater response in those with no/mild scoliosis at baseline.

Pharmacokinetic/pharmacodynamic modeling indicates that the higher dose of nusinersen may be associated with a clinically meaningful increase in efficacy above that seen with the 12-mg approved dose. Here we describe both the design of DEVOTE (NCT04089566), a 3-part clinical study evaluating safety, tolerability, and efficacy of higher dose of nusinersen, and results from the initial Part A. DEVOTE Part A evaluates safety and tolerability of a higher nusinersen dose; Part B assesses efficacy in a randomized, double-blind design; and Part C assesses safety and tolerability of participants transitioning from the 12-mg dose to higher doses. In the completed Part A of DEVOTE, all 6 enrolled participants aged 6.1-12.6 years have completed the study. Four participants experienced treatment-emergent adverse events (TEAEs), the majority of which were mild. Common TEAEs of headache, pain, chills, vomiting, and paresthesia were considered related to the lumbar puncture procedure. There were no safety concerns regarding clinical or laboratory parameters. Nusinersen levels in the cerebrospinal fluid were within the range of modeled predictions for higher dose of nusinersen. While Part A was not designed for assessing efficacy, most participants showed stabilization or improvement in motor function. Parts B and C of DEVOTE are ongoing. The findings from Part A of the DEVOTE study support further development of higher dose of nusinersen.