Neurodegenerative diseases are associated with redox stress, often linked to aberrant production of reactive species like nitric oxide (NO)1. NO can compromise TPI function via 3-Nitrotyrosination so enhancing glycation signalling, neuroinflammation, and neurodegeneration. The resulting physiological mechanisms of dysfunction are not well understood. This work uses Drosophila melanogaster to identify impacts of altered TPI activity on neuronal physiology, linking aberrant TPI function and redox stress to synaptic dysfunction at the glutamatergic Drosophila neuromuscular junction (NMJ).
Drosophila were kept at standard conditions (12hr light-dark-cycle, 25°C). TPI mutant expressing Drosophila (wstd1 and M80T point mutations2,3) were used as a disease model vs w1118 (wild-type control).
Electrophysiological recordings in two-electrode voltage-clamp were taken from muscle 6/7 in segments A2/3 of third instar larvae fillets in HL-3 buffer (1.5mM Ca2+). Evoked and spontaneous excitatory junctional currents (e/sEJCs) were recorded, alongside 60Hz train stimulations and recovery protocols to investigate synaptic depletion and subsequent recovery.
Confocal images of NMJs labelled with HRP and BRP (labelling neuronal tissue and active zone protein bruchpilot (BRP)) were taken on a Zeiss LSM 880 confocal microscope. Longevity was recorded daily.
Data is expressed as mean±SEM (n=no. of muscles). One-way ANOVA was used to test differences, longevity was tested using a Log-rank (Mantel-Cox) test, p<0.05 is significant.
Average sEJC amplitudes were -0.81±0.14nA for w1118, -0.68±0.08nA for wstd1, and -0.92±0.05nA for M80T (p>0.05), at a frequency of 1.29 ±0.18Hz for w1118, 0.83±0.08Hz for wstd1, and 1.01±0.20Hz for M80T (p>0.05, n=14, 13, 10). Average eEJC amplitudes were -95.9±5.8nA for w1118, -99.1±3.5nA for wstd1, and -88.1±8.9nA for M80T (p>0.05, n=13, 13, 10).
Synaptic depletion at 60Hz stimulation (950ms) reduced amplitudes to 41±7% of initial values in wstd1 and 57±7% in M80T vs 56±4% in w1118, exponential fits to decaying amplitudes revealed tau values of 148±28ms for wstd1 and 390±127ms for M80T vs 256±18ms for w1118 (p<0.05, n=10, 4, 9). Subsequent eEJC recovery times were altered in TPI mutants, tau: 9.2±0.8s for wstd1 and 8.2±4.4s for M80T, vs 5.8±0.6s for w1118 (p<0.05, n=10, 2, 9).
Calcium dependency of evoked release (0.25-3mM) was unaltered in wstd1 larvae compared to w1118. Confocal imaging studies did not show significant differences in NMJ morphology, bouton counts: 27.6±3.3 in w1118 and 33.0±3.8 in wstd1, active zone counts: 262±37 for w1118 and 224±20 for wstd1, active zone areas: 134±20pixels in w1118 and 95±12pixels in wstd1, and total bouton areas: 751±51pixels in w1118 and 711±131pixels in wstd1 (n=7, 10, p>0.05). TPI mutant flies showed reduced longevity, median values of 40 days in wstd1 and 42 days in M80T vs 60 days in w1118 (p<0.05, n=90, 90, 72).
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, thus expanding our knowledge of TPI involvement in synaptopathology3. Supressed TPI activity also enhances protein glycation and redox stress, which may potentially be responsible for our findings. Future studies will examine the physiological impact of redox stress, focusing on the link between NO-mediated post-translational modifications and TPI function.