Neurodegeneration has been extensively linked to aberrant production of redox active molecules, e.g. nitric oxide (NO). One target of NO-mediated post-translational modifications, specifically 3-Nitrotyrosination, is the glycolytic enzyme triose-phosphate isomerase (TPI) which catalyses the conversion of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. In parallel, reported mutations within the TPI protein render it inactive and are also associated with neurodegenerative human disease, such as in TPI deficiency which is caused by a point mutation in the TPI gene.
In this work Drosophila melanogaster expressing mutant TPI (wstd1, M80T, and I170V point mutations) were used as disease models vs wild-type controls (w1118 and Canton.S), to identify impacts of aberrant TPI function on neuronal physiology at excitatory glutamatergic neuromuscular junctions (NMJ).
Two-electrode voltage-clamp recordings were taken from third instar larvae fillets. Confocal images of larval NMJs and adult brains, labelled with HRP/BRP, and caspase/anti-AGE, were taken on a Zeiss LSM 880 confocal microscope to characterise active zones (BRP), bouton morphology (HRP), apoptosis (caspase), and advanced glycation end-products (AGE). Western blots were run as standard, longevity assays assessed daily survival.
Data is expressed as mean±SEM (n=no. of muscles/flies). Student’s t-test and Log-rank (Mantel-Cox test) were used for comparisons with p<0.05 being significant.
Longevity was seen to be reduced in TPI mutants, median lifespans of 40 and 42 days in wstd1 and M80T vs 60 and 68 days in w1118 and Canton.S respectively (p<0.005, N=145,165,146,139).
M80T showed slightly increased evoked amplitudes and Wstd1 showed significantly increased evoked amplitudes compared to Canton.S (88.12nA and 99.07nA vs 69.8nA). I170V showed significantly reduced amplitudes compared to w1118 (72.2nA vs 108.6nA).
None of the TPI mutants showed altered amplitudes of spontaneous events; 0.6787nA, 0.9194nA, 1.235nA for wstd1, M80T, and I170V, 0.8132nA and 0.8656 for w1118 and Canton. S respectively. For evoked and spontaneous events n≥9, N≥3, p<0.05.
Quantal content was seen to be reduced in I170V in comparison to w1118 (68.2 vs 183), M80T showed no significant difference and in wstd1 quantal content was seen to be increased in comparison to Canton.S (80.26, and 167.3 vs 83.93).
Synaptic depletion following 50Hz train stimulations varied between lines, amplitudes were suppressed to 67±4%, 47±6%, 52±8%, and 54±9% for w1118, wstd1, M80T, and I170V respectively (n=11,11,5,4 N=≥3 p<0.0001).
Wsdt1 and M80T lines show significantly reduced expression of TPI protein, however the I170V line does not show this reduction in comparison to controls. Preliminary confocal data suggests higher levels of AGE and apoptosis in wstd1 cf w1118. The data suggests that the TPI-mutant phenotype is in part due to altered synaptic vesicle dynamics, possibly associated with vesicle pool organisation or endo/exocytosis. Supressed TPI activity also enhances protein glycation and redox stress, possibly contributing to the observed phenotypes. Both of these possibilities offer potential therapeutic routes to manage or treat disease.