Degradation of functional triose phosphate isomerase protein underlies sugarkill pathology

Abstract

Triose phosphate isomerase (TPI) deficiency glycolytic enzymopathy is a progressive neurodegenerative condition that remains poorly understood. The disease is caused exclusively by specific missense mutations affecting the TPI protein and clinically features hemolytic anemia, adult-onset neurological impairment, degeneration, and reduced longevity. TPI has a well-characterized role in glycolysis, catalyzing the isomerization of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (G3P); however, little is known mechanistically about the pathogenesis associated with specific recessive mutations that cause progressive neurodegeneration. Here, we describe key aspects of TPI pathogenesis identified using the TPI(sugarkill) mutation, a Drosophila model of human TPI deficiency. Specifically, we demonstrate that the mutant protein is expressed, capable of forming a homodimer, and is functional. However, the mutant protein is degraded by the 20S proteasome core leading to loss-of-function pathogenesis.

Figure 1.—

Life span and behavioral analysis of animals overexpressing wild-type and mutant TPI transgenes. (A) Survival of transgenic animals at 25° overexpressing mutant and wild-type TPI. A modest reduction in longevity is observed, associated with overexpression of wild-type TPI protein. Flies overexpressing the mutant TPI, UAS-TPI^[sgk];Actin∷GAL4;+/+, do not show a significant life span deficit relative to either the control strain or animals overexpressing wild-type TPI. Longevity of TPI^sgk mutant flies is shown for comparison and is significantly reduced compared to all genotypes. N = 80–120 animals in four to six independent populations. Locomotor function in response to stress was examined in transgenic flies at 29° (B) and room temperature (C). TPI^sugarkill mutants exhibit a significantly longer recovery rate from mechanical stress compared to controls when aged at 29° (day 6) or room temperature (day 18). Transgenic overexpression of wild-type or mutant TPI protein did not result in stress-induced locomotor impairment when aged at either temperature. A total of 10–20 animals per genotype were tested. In A–C the error given is SEM and statistics were calculated using Student's t-test (**P < 0.01 and ***P < 0.001).

Figure 2.—

Size-exclusion chromatography: chromatography demonstrating that both the wild-type TPI and mutant TPI^sugarkill form a dimer. (A) HPLC chromatograph using four standards (albumin, ovalbumin, chymotrypsinogen, and ribonuclease A) to determine size retention. (B) Standard curve used to determine time of elution from the column: fraction L (>67-kDa molecules), fraction D (TPI dimer that is 53 kDa), fraction M (TPI monomer that is 26.5 kDa), and fraction C (molecules between 5 and 12 kDa). (C) Western blots using TPI antibody: each fraction from wild-type and TPI^sugarkill extracts was separated at 22° and 40°.

Figure 3.—

Western blot analysis of TPI protein levels. (A) TPI^sugarkill mutants show a reduced level of TPI protein when aged for 48 hr at room temperature and 29° compared to actin controls. (B) Quantification of Western blot shows an ∼3.5-fold decrease in TPI protein levels at room temperature and an ∼48-fold decrease at 29°. *P < 0.05 (Student's t-test). Error reported is SEM. N = 3 at each temperature. Intensity of TPI was measured from a subsaturation blot and normalized to the intensity of the actin control using ImageJ 10.2.

Figure 4.—

Survivorship of TPI^sugarkill Pros26¹ double mutants: statistical analysis of median life span of mutants aged at (A) 29° and (B) 25°. (A) Pros26¹ TPI^sgk/sgk have a modest but significant increase in longevity compared to TPI^sgk/sgk animals at 29° (P < 0.05). (B) Flies aged at 25° show a nonsignificant improvement in longevity. The median life span of Pros26¹ TPI^sgk/sgk double mutants is still much reduced from that of heterozygous controls at both temperatures examined (P < 0.0001). In A and B, the error given is SEM, statistical comparison uses Student's t-test, and n = 120 animals in six independent populations for each genotype.

Figure 5.—

Proteasome mutation increases TPI[sgk] protein levels. (A) Western blot of TPI and β-tubulin proteins from flies aged at 29° for 24 hr. Pros26¹ TPI^sgk/sgk animals show an increase in TPI protein compared to TPI^sgk/sgk mutants. (B) Quantitative analysis of TPI protein normalized to the tubulin loading control indicates a greater than twofold increase in TPI protein in the Pros26¹ TPI^sgk/sgk double mutants (*P < 0.05, n = 3 each genotype, Student's t-test). Both TPI^sgk/sgk and Pros26¹ TPI^sgk/sgk animals show a marked reduction in TPI protein when compared to wild-type controls, consistent with previous data. Error reported is SEM. Protein intensity is measured from a subsaturation image with ImageJ 10.2.

Figure 6.—

Rescue with TPI^sugarkill overexpression. Transgenic animals expressing wild-type and mutant TPI protein were examined for longevity at 25° in a TPI^sgk background. Both UAS-TPI⁺;Act∷Gal4; TPI^sgk/sgk and UAS-TPI^sgk;Act∷Gal4; TPI^sgk/sgk animals show a significant increase in life span compared to Act∷Gal4; TPI^sgk/sgk mutants (***P < 0.001, Student's t-test). Neither transgene was able to fully rescue the longevity to that of heterozygous control animals. Error given is SEM, and n = 120 animals in six independent populations.