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

Roland BP, Zeccola AM, Larsen SB, Amrich CG, Talsma AD, Stuchul KA, Heroux A, Levitan ES, VanDemark AP, Palladino MJ - PLoS Genet. (2016)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.

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.

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dTPIT73R 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 dTPIWT, dTPIT73R, dTPIT73R/Δcat and Shits1, n = 6. (E) FM1-43 unloading is unchanged between dTPIWT and dTPIT73R 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.
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pgen.1005941.g007: dTPIT73R 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 dTPIWT, dTPIT73R, dTPIT73R/Δcat and Shits1, n = 6. (E) FM1-43 unloading is unchanged between dTPIWT and dTPIT73R 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.

Mentions: We dissected larvae homozygous for dTPIWT, dTPIT73R, and Shits1 as previously detailed [46]. The NMJ preparations were heated to 38°C over 3 min. and a loading curve was generated from a series of three different high [K+] + FM1-43 loading times– 15 sec., 30 sec., and 60 sec. as previously detailed [47]. dTPIWT displays a progressive increase in dye loading from 15 sec. to 60 sec. (Fig 7A), while the temperature sensitive dynamin mutant control Shits1 showed no signs of vesicle recycling at any heated time points (Fig 7D, data not quantified). Conversely, although dTPIT73R displayed similar loading to dTPIWT at 15 and 30 sec., dTPIT73R exhibited a striking 50% decrease in loading at 60 sec. (Fig 7A, 7B and 7D). This progressive decrease in endocytosis was stimulation and temperature dependent; loading experiments performed at room temperature did not exhibit an endocytic defect (Fig 7C). As previous experiments had highlighted the capacity of dTPIΔcat to complement the adult behavioral defects of dTPIT73R, we examined dTPIT73R/dTPIΔcat larvae to assess the relationship between vesicle endocytosis and animal behavior. The dTPIT73R/dTPIΔcat animals displayed a significant increase in vesicle endocytosis relative to dTPIT73R (Fig 7B and 7D). These results demonstrate that dTPIΔcat complements adult behavior and vesicle endocytosis defects. The utilization of chemical stimulation in these preparations demonstrates a synaptic defect arising from the severe dTPIT73R dimer mutation as this methodology bypasses conductance requirements.


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

Roland BP, Zeccola AM, Larsen SB, Amrich CG, Talsma AD, Stuchul KA, Heroux A, Levitan ES, VanDemark AP, Palladino MJ - PLoS Genet. (2016)

dTPIT73R 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 dTPIWT, dTPIT73R, dTPIT73R/Δcat and Shits1, n = 6. (E) FM1-43 unloading is unchanged between dTPIWT and dTPIT73R 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4816394&req=5

pgen.1005941.g007: dTPIT73R 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 dTPIWT, dTPIT73R, dTPIT73R/Δcat and Shits1, n = 6. (E) FM1-43 unloading is unchanged between dTPIWT and dTPIT73R 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.
Mentions: We dissected larvae homozygous for dTPIWT, dTPIT73R, and Shits1 as previously detailed [46]. The NMJ preparations were heated to 38°C over 3 min. and a loading curve was generated from a series of three different high [K+] + FM1-43 loading times– 15 sec., 30 sec., and 60 sec. as previously detailed [47]. dTPIWT displays a progressive increase in dye loading from 15 sec. to 60 sec. (Fig 7A), while the temperature sensitive dynamin mutant control Shits1 showed no signs of vesicle recycling at any heated time points (Fig 7D, data not quantified). Conversely, although dTPIT73R displayed similar loading to dTPIWT at 15 and 30 sec., dTPIT73R exhibited a striking 50% decrease in loading at 60 sec. (Fig 7A, 7B and 7D). This progressive decrease in endocytosis was stimulation and temperature dependent; loading experiments performed at room temperature did not exhibit an endocytic defect (Fig 7C). As previous experiments had highlighted the capacity of dTPIΔcat to complement the adult behavioral defects of dTPIT73R, we examined dTPIT73R/dTPIΔcat larvae to assess the relationship between vesicle endocytosis and animal behavior. The dTPIT73R/dTPIΔcat animals displayed a significant increase in vesicle endocytosis relative to dTPIT73R (Fig 7B and 7D). These results demonstrate that dTPIΔcat complements adult behavior and vesicle endocytosis defects. The utilization of chemical stimulation in these preparations demonstrates a synaptic defect arising from the severe dTPIT73R dimer mutation as this methodology bypasses conductance requirements.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.

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.

Show MeSH
Related in: MedlinePlus