The expression of Hsap\MAPTUAS.cWa under the control of Scer\GAL4GMR.PF induces a small and rough eye phenotype, with ommatidial irregularity, as compared to controls. Primary neuron cultures from their third instar larval brains show a significant reduction in the average length of the neural extensions compared to controls.
The expression of Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4elav-C155 leads to a significant and progressive decrease in adult locomotor activity (in negative geotaxis assays), but does not significantly affect the number of cortical neurons in the anterior medulla, as compared to controls.
Expression of Hsap\MAPTScer\UAS.cWa driven by Scer\GAL4ninaE.PT (crosses carried out at 18[o]C and shifted to 25[o]C following eclosion) does not significantly affect external eye morphology in adult flies. At 1 day old, electroretinogram (ERG) recordings of light induced potentials from Scer\GAL4ninaE.PT>Hsap\MAPTScer\UAS.cWa flies are similar to controls; aged flies (day 10 and day 30) show a significant loss of light-evoked ERG response (significant reduction in depolarization amplitude) compared to controls. Scer\GAL4ninaE.PT>Hsap\MAPTScer\UAS.cWa flies do not show significant differences to controls in terms of ON transient amplitude; OFF transient amplitudes are significantly increased at day 1 but significantly decreased at day 10 and 30.
The number and morphology of photoreceptors (retinae show intact ommatidia with 7 visible rhabdomeres) in Scer\GAL4ninaE.PT>Hsap\MAPTScer\UAS.cWa flies is grossly preserved at all time points, similar to controls; however photoreceptor ultrastructure (examined with TEM) in 10 day old Scer\GAL4ninaE.PT>Hsap\MAPTScer\UAS.cWa flies revealed significant subtle defects and irregularities in rhabdomeres (splitting or fraying at edges, vacuolar changes). Mutants at day 10 have similar numbers of mitochondria per ommatidium and numbers of autophagic vacuoles as controls, but have a significant increase in the percentage of photoreceptors with electron dense vacuoles (telolysosomes). Scer\GAL4ninaE.PT>Hsap\MAPTScer\UAS.cWa flies show significant disruption of photoreceptor synaptic terminal organization and morphology at 10 days old compared to controls.
Limited degeneration is seen in a small subset of PPL1 dopaminergic neurons in 2 week old adults expressing Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4ple.PF. At 4 weeks after eclosion a significant loss of both PPL1 and PPM3 dopaminergic neurons is seen.
Flies expressing Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4ple.PF show an age-dependent climbing deficit, with a significant loss of climbing ability compared to controls at 4-6 weeks post-eclosion. 4 week old flies also show reduced spontaneous locomotion compared to controls, appearing sluggish with an increase in the number of pauses between bouts of spontaneous locomotion and travelling short distances during each bout.
3 week old flies expressing Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4ple.PF show a modest but significant learning defect compared to control flies in an olfactory associative learning assay.
4 week old flies expressing Hsap\MAPTScer\UAS.cWa under the control of either Scer\GAL4VGlut.PD or Scer\GAL4Gad1.3.098 do not show a loss of Hsap\MAPTScer\UAS.cWa expressing cells in the brain (glutamatergic or GABAergic cells respectively).
4 week old flies expressing Hsap\MAPTScer\UAS.cWa under the control of either Scer\GAL4Hn.996 or Scer\GAL4npf.1 show a loss of the cells expressing Hsap\MAPTScer\UAS.cWa compared to wild type.
Expression of Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4C57 results in microtubule fragmentation and reduced perinuclear microtubule density in the muscles.
Expression of Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4elav.PU results in a significant increase in the number of satellite boutons at the neuromuscular junction (NMJ) and a significant decrease in average NMJ length compared to controls.
Flies expressing Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4elav.PU exhibit normal learning and memory in an aversive phototaxis assay at two days after eclosion. Learning and memory are both significantly reduced at 20 days post-eclosion.
Expression of Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4elav.PU results in an age-dependent decrease in locomotor activity as measured by climbing performance.
At two days post eclosion no vacuolisation is observed in the brains of flies expressing Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4elav.PU. By 20 days multiple large vacuoles can be observed.
The mitochondria in the neurons of adult brains expressing Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4elav.PU are markedly elongated. Mitochondria length is on average greater than twice that of controls.
Expression of Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4GMR.PF causes age-dependent and progressive neurodegeneration in the lamina; degeneration in the lamina is undetectable or very mild in flies at 3-day old, but is prominent at 10 days old. As in wild type, mitochondria are present in the synaptic terminals of photoreceptor neurons at 3 days old.
3 day old flies expressing Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4GMR.PF show display axon pathology in the lamina. This includes the formation of vacuoles in the axons and swollen axons. In the presynaptic terminals, vacuoles and the accumulation of autophagic bodies and multivesicular bodies are seen.
Expression of Hsap\MAPTScer\UAS.cWa in the developing retina, under the control of Scer\GAL4GMR.PF, causes a moderate rough eye phenotype, characterised by disruption of the ordered ommatidial arrangement and a moderate reduction in the size of the eye.
Overexpression of Hsap\MAPTUAS.cWa under the control of Scer\GAL4RapGAP1-OK6 in larval segmental nerves effects synaptic vesicle kinetics (there is a significant increase in synaptic vesicle pausing time, but no significant change on the directionality of the vesicular transport); there is no significant reduction in MT density; no effect on the number of mobile vesicles within axons. Emerging flies have normal wing expansion.
Expression of Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4repo.PU is lethal unless the transgene expression is temporally limited by combination with tub-Gal80[ts] (and accompanying culturing temperature manipulations) to minimize expression during development, in which case adult flies eclose but show significantly decreased lifespan and age-progressive increase in the number of apoptotic glia as well as cholinergic neurons (non-cell-autonomous effect) in the brain. Fibrillary tangles of phosphorylated tau protein are observed in the proximal as well as distal processes of glial cells in aged flies. Rearing the eclosed adults at 30[o]C for 20 days (to switch on Hsap\MAPTScer\UAS.cWa expression) and then moving them to 17[o]C for 10 days (to switch off expression) significantly reduces the number of apoptotic cells but does not decrease the number of the fibrillary inclusions in the brain.
Expression of Hsap\MAPTScer\UAS.cWa under the control Scer\GAL4elav.PU (with tub-Gal80[ts] to limit time of expression to the adult stage) also results in age-progressive increase in the number of apoptotic cells in the adult brain and this increase is further enhanced when both the Scer\GAL4repo.PU and the Scer\GAL4elav.PU driver are used together: the number of apoptotic cells is much higher than when either of the two drivers is used alone.
Expression of Hsap\MAPTScer\UAS.cWa driven by Scer\GAL4Appl.G1a induces substantial axonal vesicle accumulations in larvae and results in increased lethality during development, as compared to controls.
Although the overall structure and morphology of the adult brains expressing Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4elav.PU appear unaltered, the mushroom bodies (MBs) are severely reduced or entirely absent in the majority of animals. The dendrites of MBs are prominent in controls, but not apparent in Hsap\MAPTScer\UAS.cWa-expressing animals. Other neuropils such as the protocerebral bridge in the posterior of the head and the fan-shaped body appear normal.
Pan-neuronal expression of Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4elav.PU results in the loss of Kenyon cells in third-instar larval brains, whereas neurons in other parts of the central nervous system appear unaffected.
MB defects are observed in 75% of the adults expressing Hsap\MAPTScer\UAS.cWa conditionally throughout development on to adulthood under the control of Scer\GAL4elav.PU and Scer\GAL80ts.αTub84B. Similarly, 70% of adults expressing Hsap\MAPTScer\UAS.cWa exclusively through embryogenesis display MB defects. In contrast, conditional expression of Hsap\MAPTScer\UAS.cWa after hatching, from early larva to adulthood results in mild MB defects in less than 5% of animals examined. Transgene expression exclusively in pupae does not result in detectable defects, and the effects of raising transgene-harboring animals at the restrictive temperature are negligible.
Temperature shift experiments reveal that conditional expression of Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4elav.PU and Scer\GAL80ts.αTub84B during the first 4 h of embryogenesis at 19[o]C does not affect adult MB structure. In contrast, continued expression of Hsap\MAPTScer\UAS.cWa 8 h into embryogenesis results in MB abnormalities in adults. Continued expression of Hsap\MAPTScer\UAS.cWa up to 14 h after egg laying does not result in more severe deficits.
Compared with control embryos, cells at the stereotypical mushroom body neuro-ectoderm that are known to give rise to mushroom body neuroblasts in the head of stage 9-12 Scer\GAL4elav.PU>Hsap\MAPTScer\UAS.cWa-expressing embryos are not apparent.
When Hsap\MAPTScer\UAS.cWa is expressed in mushroom body neuroblasts and their progeny under the control of Scer\GAL4ey-OK107, animals with obvious MB deficits are obtained.
Expression of Hsap\MAPTScer\UAS.cWa limited to the ellipsoid body under the control of Scer\GAL4Aph-4-c232 is not toxic for MB neurons.
Expression with Scer\GAL4ey.PU causes a rough eye phenotype, which results in reduction of the eye tissue in extreme cases.
Expression of Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4elav.PU results in an increase in the number of apoptotic neurons in the brains of 10-day old flies, as compared to controls.
Expression of Scer\GAL4ey.PB>Hsap\MAPTScer\UAS.cWa leads to three different classes of phenotype: normal-looking eyes, abnormal eyes (characterised by abnormal shape or rough surface) and loss of eyes (no eyes). Rapamycin treatment reduces the proportion of flies without eyes, whereas increasing the proportion of flies with normal eyes. Rapamycin also increases the survival of flies expressing Hsap\MAPTScer\UAS.cWa to the adult stage.
Expression of Hsap\MAPTScer\UAS.cWa in the adult mushroom body and other brain neurons under the control of either Scer\GAL4c492b or Scer\GAL4c747 does not result in decreased longevity. Hsap\MAPTScer\UAS.cWa-expression does not appear to cause gross morphological abnormalities or decreased fecundity and vigor compared with controls. Degenerating cells and large vacuoles are not detected in the mushroom bodies of controls or 21 day old animals expressing Hsap\MAPTScer\UAS.cWa.
21 day old flies expressing Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4c492b do not exhibit obvious degeneration or vacuoles in the mushroom body. Mild neurodegeneration is observed in the mushroom bodies of most 60 day old flies expressing Hsap\MAPTScer\UAS.cWa using Scer\GAL4c492b. Degenerating neurons are observed in the mushroom body, but not in the ellipsoid body where the Scer\GAL4c492b driver is inactive.
The performance of flies expressing Hsap\MAPTScer\UAS.cWa under the control of either Scer\GAL4c492b or Scer\GAL4c747 is not significantly different from controls in negatively reinforced associative olfactory learning tasks.
The avoidance-response to electrified grids kept at 90 V or 15 V is not different between animals expressing Hsap\MAPTScer\UAS.cWa under the control of either Scer\GAL4c492b or Scer\GAL4c747 and controls. Hsap\MAPTScer\UAS.cWa-expressing animals and controls exhibit equal decrements in response to olfactory conditioned stimuli after experiencing couples conditioned plus unconditioned stimuli.
Scer\GAL4c747>Hsap\MAPTScer\UAS.cWa flies exhibit a highly significant impairment in learning compared with controls. The memory of the conditioned association 90 minutes later is significantly depressed in Hsap\MAPTScer\UAS.cWa-expressing animals, and to a lesser degree at 180 minutes.
Conditioned/unconditioned pairing in Scer\GAL4c747>Hsap\MAPTScer\UAS.cWa-expressing flies yields olfactory learning scores nearly 50% lower than those of controls. The Hsap\MAPTScer\UAS.cWa-expressing animals exhibit a significant decrease in memory retention after 30 minutes, despite performing at equivalent level with controls immediately after training.
Expression of Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4elav.PLu results in a moderate reduction in adult life span. The effect on life span is dosage sensitive. Aged flies show neurodegeneration; vacuolisation and degeneration of cells in the cortex is seen. The degeneration is progressive and the phenotype is fully penetrant. No evidence of large filamentous aggregates (neurofibrillary tangles) is seen in the brains of adults expressing Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4elav.PLu or Scer\GAL4Cha.7.4. Progressive loss of cholinergic neurons in the optic lamina is seen in adults expressing Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4Cha.7.4. 1 day old adults expressing Hsap\MAPTScer\UAS.cWa under the control of Scer\GAL4elav.PLu have a normal number of cholinergic terminals in the brain. The number of acetylcholine-positive terminals is strongly reduced in aged flies.