A16269478G
A?G
N262S | Khc-PA
N262S
Analogous mutation in human KIF5A implicated in spastic paraplegia 10; mutation carried on in vitro construct.
Larvae expressing KhcN262S.Scer\UAS under the control of Scer\GAL4D42 at either 25 or 29[o]C initially display a "tail-flipping" phenotype, and the paralysis then ascends from posterior to anterior until the larvae can only move their head. Complete paralysis then ensues, with lethality in the second or third instar. Larval growth is delayed compared to wild type. Before paralysis, larval locomotion speed is reduced compared to wild type. Axonal swellings are seen in the mutant larvae. These swellings are filled with mitochondria, pre-lysosomal vacuoles, autophagosomes and multivesicular bodies.
A strong reduction in both anterograde and retrograde flux of mitochondria along the axons is seen in larvae expressing KhcN262S.Scer\UAS under the control of Scer\GAL4D42.
Larvae expressing KhcN262S.Scer\UAS under the control of Scer\GAL4D42 show a strong reduction in the number and density of mitochondria at neuromuscular junctions (NMJs) 6/7 in segment A2, while there is no obvious reduction in mitochondrial abundance in motorneuron cell bodies. The area of NMJ 6/7 is strongly reduced compared to controls in segment A5 but not in segment A2. The number of synapses at the NMJ in segment A2 is not significantly different from wild type, while the number of synapses at the NMJ in segment A5 is significantly reduced. A significant degree of neurodegenerative alterations are seen at NMJ 6/7 of segment A5 in mutant larvae.
No evidence for increased apoptosis of motorneurons is seen in mid-third instar larvae expressing KhcN262S.Scer\UAS under the control of Scer\GAL4D42.
The evoked excitatory junctional potential (EJP) of the NMJ is drastically reduced in amplitude in larvae expressing KhcN262S.Scer\UAS under the control of Scer\GAL4D42, while the half-width time is increased. The amplitude of the miniature EJP is slightly, but not significantly, increased in the mutant larvae. Quantal content is significantly reduced.
Adults with a 25% reduction in lifespan are recovered when KhcN262S.Scer\UAS is expressed under the control of Scer\GAL4D42 at 18[o]C. These animals cannot sustain stable flight if forced to fly, but fall to the ground. At rest they have a held-up wing phenotype. No evidence of degeneration of the indirect flight muscles can be seen.
16 day old adults in which KhcN262S.Scer\UAS is expressed under the control of Scer\GAL4elav.PU after eclosion (the temperature sensitive Scer\GAL80ts.αTub84B allele is used to limit expression prior to eclosion) are essentially unable to climb.
A strong reduction in both anterograde and retrograde flux of mitochondria along the axons is seen in larvae co-expressing both KhcScer\UAS.cFa and KhcN262S.Scer\UAS under the control of Scer\GAL4D42.
Quantal content at the neuromuscular junction is significantly reduced in larvae co-expressing both KhcScer\UAS.cFa and KhcN262S.Scer\UAS under the control of Scer\GAL4D42.
KhcN262S.UAS is partially rescued by KhcUAS.cFa/Scer\GAL4Toll-6-D42
Co-expression of KhcScer\UAS.cFa ameliorates the paralysis phenotype seen in larvae expressing KhcN262S.Scer\UAS under the control of Scer\GAL4D42 at either 25 or 29[o]C , with the larvae displaying a tail-flipping phenotype at a stage where larvae expressing only KhcN262S.Scer\UAS under the control of Scer\GAL4D42 can only move their heads.
