Structural and Genetic Studies Demonstrate Neurologic Dysfunction in Triosephosphate Isomerase Deficiency Is Associated with Impaired Synaptic Vesicle Dynamics

Abstract

Triosephosphate isomerase (TPI) deficiency is a poorly understood disease characterized by hemolytic anemia, cardiomyopathy, neurologic dysfunction, and early death. TPI deficiency is one of a group of diseases known as glycolytic enzymopathies, but is unique for its severe patient neuropathology and early mortality. The disease is caused by missense mutations and dysfunction in the glycolytic enzyme, TPI. Previous studies have detailed structural and catalytic changes elicited by disease-associated TPI substitutions, and samples of patient erythrocytes have yielded insight into patient hemolytic anemia; however, the neuropathophysiology of this disease remains a mystery. This study combines structural, biochemical, and genetic approaches to demonstrate that perturbations of the TPI dimer interface are sufficient to elicit TPI deficiency neuropathogenesis. The present study demonstrates that neurologic dysfunction resulting from TPI deficiency is characterized by synaptic vesicle dysfunction, and can be attenuated with catalytically inactive TPI. Collectively, our findings are the first to identify, to our knowledge, a functional synaptic defect in TPI deficiency derived from molecular changes in the TPI dimer interface.

Fig 1. hTPI^M82T elicits a conformational change in TPI resulting in reduced dimerization.

(A) Sequence alignment of D.melanogaster and H.sapiens TPI protein sequence with asterisks highlighting residues of interest. (B) The hTPI^M82T mutation confers a reduction in mean protein hydrodynamic radius as measured by dynamic light scattering. (C) Intensity correlation plots reveal a largely monodisperse hTPI^WT population and polydisperse hTPI^M82T population. (D) Gel filtration indicates a change in monomer:dimer ratios elicited by hTPI^M82T with relative quantification (inset). n≥3, comparisons were made using Student’s T test, *** indicates p<0.001.

Fig 2. Mutations affecting the TPI dimer interface recapitulate dTPI^M80T phenotypes.

(A) dTPI^T73R and dTPI^G74E homozygotes display severely reduced lifespans, n>150. (B) Dimer interface mutations exhibit severe mechanical stress at Day 1 and (C) thermal stress sensitivity at Day 2, n>30. Thermal stress paralysis times at 360 sec. represent wild type behavior, the assay was stopped at 6 min. (D) Both dTPI^T73R and dTPI^G74E homozygotes display reduced lysate isomerase activity, n≥3. Comparisons were made with a One-way ANOVA using Tukey’s post hoc test, and lifespans by a Log-rank (Mantel-Cox) survival test, ** indicated p<0.01, *** p<0.001.

Fig 3. Mutations at the TPI dimer interface reduce TPI levels in vivo without aggregation.

(A) dTPI^T73R and dTPI^G74E homozygote animal lysates display reduced protein levels with (B) quantification normalized to WT and an ATPalpha loading control, n = 3. (C) The reduction in SDS-soluble TPI is not caused by protein aggregation; increasing amounts of lysate were loaded and show no differences in filter-trapped TPI across all genotypes, (D) 10μg of huntingtin exon1-GFP lysate displayed robust retention on the filter, n = 2. Comparisons were made with a One-way ANOVA using Tukey’s post hoc test, *** p<0.001 relative to WT.

Fig 4. A catalytically inactive allele attenuates TPI dimer mutant behavior and longevity.

(A) dTPI^Δcat complements dTPI^T73R mechanical and (B) thermal stress sensitivity and (C) longevity, and (D,E) partially attenuates behavior and (F) longevity defects of dTPI^G74E. Thermal stress paralysis times at 360 sec. represent wild type behavior, the assay was stopped at 6 min. n≥30 for behavior, and n≥90 for lifespans. Behavior was compared using a One-way ANOVA with Tukey’s post hoc test, and lifespans by a Log-rank (Mantel-Cox) survival test, ns indicates no significance, and *** p<0.001.

Fig 5. hTPI^Δcat crystal structure reveals a catalytically incompetent enzyme with an unaltered dimer interface.

(A) The hTPI^Δcat adopts an open lid conformation. Superposition of hTPI^WT (grey) and hTPI^Δcat (blue) structures. Loop3 (tan), the position of the lid covering the TPI active site (magenta and pink), and the locations of bromide and phosphate ions from the active site pocket of hTPI^WT are indicated. New hydrophobic interactions that help to reposition M13 are shown (green). (B) The dimer interface of hTPI^Δcat is unchanged relative to hTPI^WT. Superposition of hTPI^WT (grey) and hTPI^Δcat shown as in (A), with monomer subunits of hTPI^Δcat dimer in blue and tan. Key dimer interface residues and loops are indicated.

Fig 6. Heterodimerization of inactive TPI and dimer interface mutations.

(A) dTPI^Δcat-CFP interacts modestly with dTPI^M80T, dTPI^T73R, and dTPI^T73R,G74E, yet robustly with dTPI^G74E. Representative immunoprecipitation and input blots are shown with (B) IP:anti-GFP quantification n = 3. Quantification represents 25kD TPI IP signal, with negative control subtracted, normalized to the lysate β-tubulin loading control, and compared to WT. Comparisons were made with a One-way ANOVA using Tukey’s post hoc test, ns indicates no significance, ** p<0.01, and *** p<0.001.

Fig 7. dTPI^T73R impairs NMJ synaptic vesicle dynamics.

(A) An FM1-43 timecourse at the NMJ with loading times of 15, 30, and 60 sec., (B) with quantification of 60 sec. at 38°C, and (C) 60 sec. at room temperature (RT). (D) Representative images of dTPI^WT, dTPI^T73R, dTPI^T73R/Δcat and Shi^ts1, n = 6. (E) FM1-43 unloading is unchanged between dTPI^WT and dTPI^T73R at 38°C with animal replicates indicated, and (F) representative images. Comparisons were made with a One-way ANOVA using Tukey’s post hoc test, ***p<0.001. Scale bars = 5μm.

Fig 8. TPI dimer interface substitutions do not alter NMJ development and morphology.

(A) NMJ morphology of segment A2 muscle 6/7 was characterized for (B) bouton number and (C) branching. Boutons were defined as varicosities greater than 2 μm in diameter. Neither parameter showed significant differences elicited by the mutations, relative to either dTPI^WT or dTPI^WT-CFP/TPI^WT. CFP tags did not alter the behavioral deficits of the animals (S6 Fig). n = 10. Comparisons were made with a One-way ANOVA using Tukey’s post hoc test, ns indicated no significance. Scale bar = 10 μm.

Fig 9. hTPI^Δcat models predict that hTPI^Δcat::hTPI^G76E heterodimerization alters the TPI dimer interface.

(A) The R75 side chain may insert itself into the active site in the context of a hTPI^Δcat::hTPI^T75R heterodimer. Shown is a superposition of hTPI^WT (grey), hTPI^Δcat (blue), and hTPI^Δcat::hTPI^T75R (green) obtained from Rosetta Backrub modeling. Loop3, containing T75R and G76E, is indicated in tan. For clarity, the mainchain of hTPI^Δcat::hTPI^T75R has been omitted. (B) Repositioning of E104 and R98 side chains assists the dimer interface to accommodate the G76E substitution in Rosetta modeling. Superposition of hTPI^Δcat structure (blue) with the hTPI^Δcat::hTPI^G76E model (green). In both structures, Loop 3 is shown in tan and selected active site pocket residues are indicated. (C) Modeling the effect of T75R and G76E substitutions in the context of hTPI^WT and hTPI^Δcat structures. Normalized scores for the best 10% of 50 simulations were averaged for each experimental structure with the indicated computational substitution. Higher scores indicate a resulting model that is less favorable using the Rosetta energy function.