Introduction Alzheimer’s disease (AD) patients exhibit disturbed daily sleep-wake patterns (1). We have used a transgenic fruit fly model expressing aggregation-prone versus control forms of the amyloid beta (Abeta = Aβ) peptide which constitutes the plaques in AD brain tissue to investigate the molecular basis of disturbed sleep-wake cycles in AD. Here we examined the circadian locomotor activity of flies expressing the disease-linked aggregation prone Aβ isoforms Aβ1-42 and the arctic (E22G) variant of Aβ1-42 (Aβ1-42Arc) during the first 15 days of adult life. These flies, which exhibit a range of AD-related phenotypes, were compared to control flies expressing the less-aggregation-prone peptide Aβ1-40, un-activated transgenic lines, the background strain 51D for the transgenic lines and the elav driver strain for transgene expression.  

Materials and methodsSUBJECTS:

Transgenic flies (2) expressed the Beta-amyloid peptides when crossed with the elav pan-neuronal driver strain. The driver strain’s transcription factor activates the yeast UAS-GAL4 promoter in the offspring of elav x transgenic line parents. Background strain 51D flies were used as wild-type controls. The transgenes are all inserted at the same locus. 51D and the three transgenic Aβ lines were also assayed for Aβ42ARC expression with a tim(62)-gal4 driver specific to clock neurons.

GENOTYPES TESTED:

51D : Wild Type Background StrainAb40-51D: Ab40 Isoform on 51D BackgroundAb42-51D: Ab42 Isoform on 51D BackgroundAb42arc51D: Ab42Arc Isoform on 51D BkgroundElav: Transgene driver strainElav x 51D: Elav driving controlElav x Ab40: Elav driving Ab40 ExpressionElav x Ab42: Elav driving Ab42 ExpressionElav x Ab42Arc: Elav driving Ab42arc Expression

KEY:51D: Wild Type Background Strain elav: Transgene Driver StrainAb40: Low Aggregation Aβ PeptideAb42: Aggregation-Prone Aβ Peptide Ab42Arc: Severe Aggregation-Prone Aβ Peptide

RESULTS

Free Running Period (tauDD):

Circadian period was significantly different among the crosses (Fig 2).

Conclusions

OBJECTIVE:Effects of Aβ peptides (Aβ40, Aβ42 and Aβ42arc) on circadian locomotor activity and sleep were examined in a Drosophila model for Alzheimer’s disease.

CIRCADIAN PERIOD:Overall, the circadian period of all three transgenic lines shortened when Aβ peptide transgene expression was induced by the elav driver strain. The 51D background control strain for the transgenic lines did not change significantly when crossed with elav. This trend was statistically significant for the Aβ40 peptide, but it is difficult to separate from the effect of crossing the transgenic lines with the short-period elav driver and requires further investigation.

CIRCADIAN AMPLITUDE:The Aβ42arc transgenic strain displayed the lowest amplitude circadian rhythms and the greatest proportion of arrhythmic flies in the unactivated transgenic lines. This effect was amplified in older flies. It only occurred with the pan-elav driver, and was not observed when Aβ42arc expression was induced with a clock-cell-specific tim(62)-gal4 driver, indicating that the mechanism involved is downstream from the central clock. It would be useful to examine output pathways for activity, sleep and additional rhythms.

SLEEP:Mean sleep and LD activity scores were generally similar among all Aβ transgenic lines with 51D and elav at the extremes. Sleep in the ARC and ARC x elav strains, and In 51D x elav was most fragmented. This observation is consistent with the effect of Aβ42arc expression on the rhythmicity of locomotor activity.

Do Aβ Peptides Alter Circadian Locomotor Behaviour in a Transgenic Drosophila Model of Alzheimer’s Disease?

Chen K, Possidente B, Sleezer B, Clinton S, Sheppard A D Crowther Cambridge University, Dept. of Genetics, Cambridge UK Skidmore College, Saratoga Springs, NY Dept. of Biology USA SUNY Albany Albany, NY Dept. of Biology USA

Figure 1. Drosophila Activity Monitor (TriKinetics Inc., Waltham, MA). Each monitor records activity from 32 individual flies.

CIRCADIAN BEHAVIOR ASSAY

Male flies aged 1-2 days post-eclosion were assayed for circadian locomotor behavior and sleep for 3-5 days in 12:12 LD followed by 4-6 days in DD in Trikinetics Drosophila Activity Monitors (Fig 1) . Two replicates, one at Cambridge University, UK and one at Skidmore College, USA were assayed and pooled for a total sample size of approximately 35 flies per genotype. A set of older flies aged 10-20 days was also assayed at Cambridge University.

STATISTICAL ANALYSIS:

Circadian period was estimated using chi-squared periodogram, autocorrelation and MESA . A rhythmicity score (RS) was derived from the autocorrelation estimate (4) to evaluate amplitude. Strain differences were tested with ANOVA and Bonferroni post-hoc comparisons.

Fig 2. Background 51D and unactivated transgenic lines had slightly longer tauDD compared to elav-activated lines with Elav showing the shortest tauDD. Post-hoc tests showed elav x Ab40 to be significantly shorter than elav x 51D. These results suggest that expression of the Aβ40 peptide may shorten tauDD.

Post-hoc

P <.0001 All Groups

Fig 3. Ab42arc flies, whether activated with elav or not, had the lowest proportion of rhythmic flies (Fig 3, left) and the lowest amplitude rhythms among rhythmic flies (Fig 3, middle). Post-hoc tests showed a significant difference between the elav x Ab40 and the elav x Ab42arc lines in young (middle) and older (right panel) flies, plus elav x AB42arc vs. elav x 51D in older flies (right panel). This result suggests that expression of the ab42arc Aβ peptide weakens circadian rhythm amplitude, the effect increases with age, and that the ab42arc peptide is expressed to some degree in the un-activated transgenic strain (3).

RHYTHMICITY

P <0.03 All Groups

Post-hoc

ACTOGRAMS: Composite actograms for each genotype,with two days in 12:12 LD followed by six days in DD, are shown for the flies tested at Skidmore College. Taudd isSignificantly shorter in AB40 x elav compared to 51D x elav, and both the transgenic and activated ab42arc lines have lower amplitude rhythms.

LITERATURE CITED1. Thome J, Coogan AN, Woods AG, Darie CC, Häßler F (2011) CLOCK Genes and

Circadian Rhythmicity in Alzheimer Disease. J Aging Res 201:383091 Epub Oct 18.2. Stocks provided by Damian Crowther, Department of Genetics, Cambridge University, UK.3. http://www.frontiers-in-genetics.org/flyc31/starter_kit.php4. Levine JD, Funes P, Dowse HB, Hall JC (2002) Signal analysis of behavioral and molecular cycles. BMC Neuroscience 3:1.

51D AB40 AB42 ARC

51D x elav AB40 x elav AB42 x elav ARC x elav

elav

taudd=23.30

taudd=23.45

taudd=24.05

taudd=23.45

taudd=24.05

taudd=23.25

taudd=23.55taudd=24.00

taudd=23.50

SLEEP AND ACTIVITY SCORE: SLEEP, SLEEP BOUT AND LD ACTIVITY SCORES

GENOTYPE

51D AB40 AB42 ARC elav el-51D el-AB40el-AB42 el-ARC

SC

OR

E

0

20

40

60

80

100

120

SLEEP SCORE (INACTIVE 10/MIN BINS/DAY)SLEEP FRAGMENTATION SCORE (BOUTS/DAY)ACTIVITY IN LD (COUNTS/10MIN)

Sleep (p<.0001; number of inactive 10/min bins/day), sleep bout number per day (p<.003) and mean activity counts per 10mins in 12:12 LD (p<.0001) were significantly different among genotypes. Activity and sleep were inversely associated, with 51D and elav at the extremes and transgenic lines in between. ARC, ARC x elav and 51D x elav displayed the most fragmented sleep.Means +/- SEM are shown.

----------Age 3-9 Days---------- Age 10-20 Days

El-51D el-AB40 el-AB42 el-ARC

1) tim(62) > 51D 2) tim(62) > A40 3)tim(62) > A42 4) tim(62) > A42arctic

ACTOGRAMS FOR OLDER FLIES (10-20 Days): Decreased rhythmicity in Aβ42arc flies is enhanced in older flies.

ACTOGRAMS FOR tim(62)-gal4 EXPRESSION IN CLOCK NEURONS: Expression of Aβ peptides limited to clock neurons did not alter rhythmicity, indicating that the effect of Aβ42arc on rhythmicity is downstream from the clock mechanism.

51D x elav AB40 x elav AB42 x elav ARC x elav

n.s.

El-51D el-AB40 el-AB42 el-ARC