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

<div><p>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.</p></div

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

<p>(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 <i>dTPI</i>^<i>WT</i> or <i>dTPI</i>^{<i>WT-CFP</i>}<i>/TPI</i>^<i>WT</i>. CFP tags did not alter the behavioral deficits of the animals (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005941#pgen.1005941.s006" target="_blank">S6 Fig</a>). n = 10. Comparisons were made with a One-way ANOVA using Tukey’s post hoc test, <i>ns</i> indicated no significance. Scale bar = 10 μm.</p

Heterodimerization of inactive TPI and dimer interface mutations.

<p>(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, <i>ns</i> indicates no significance, ** p<0.01, and *** p<0.001.</p

<i>dTPI</i>^<i>T73R</i> impairs NMJ synaptic vesicle dynamics.

<p>(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 <i>dTPI</i>^<i>WT</i>, <i>dTPI</i>^<i>T73R</i>, <i>dTPI</i>^{<i>T73R/Δcat</i>} and <i>Shi</i>^<i>ts1</i>, n = 6. (E) FM1-43 unloading is unchanged between <i>dTPI</i>^<i>WT</i> and <i>dTPI</i>^<i>T73R</i> 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.</p

Mutations at the TPI dimer interface reduce TPI levels <i>in vivo</i> without aggregation.

<p>(A) <i>dTPI</i>^<i>T73R</i> and <i>dTPI</i>^<i>G74E</i> homozygote animal lysates display reduced protein levels with (B<b>)</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.</p

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

<p>(A) Sequence alignment of <i>D</i>.<i>melanogaster</i> and <i>H</i>.<i>sapiens</i> 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.</p

Mutations affecting the TPI dimer interface recapitulate <i>dTPI</i>^<i>M80T</i> phenotypes.

<p>(A) <i>dTPI</i>^<i>T73R</i> and <i>dTPI</i>^<i>G74E</i> 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 <i>dTPI</i>^<i>T73R</i> and <i>dTPI</i>^<i>G74E</i> 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.</p

Crystallographic Data collection and Refinement statistics.

<p>Crystallographic Data collection and Refinement statistics.</p

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

<p>(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.</p