Oscillating PDF in termini of circadian pacemaker neurons and synchronous molecular clocks in downstream neurons are not sufficient for sustenance of activity rhythms in constant darkness

Abstract

In Drosophila, neuropeptide Pigment Dispersing Factor (PDF) is expressed in small and large ventral Lateral Neurons (sLNv and lLNv), among which sLNv are critical for activity rhythms in constant darkness. Studies show that this is mediated by rhythmic accumulation and likely secretion of PDF from sLNv dorsal projections, which in turn synchronises molecular oscillations in downstream circadian neurons. Using targeted expression of a neurodegenerative protein Huntingtin in LNv, we evoke a selective loss of neuropeptide PDF and clock protein PERIOD from sLNv soma. However, PDF is not lost from sLNv dorsal projections and lLNv. These flies are behaviourally arrhythmic in constant darkness despite persistence of PDF oscillations in sLNv dorsal projections and synchronous PERIOD oscillations in downstream circadian neurons. We find that PDF oscillations in sLNv dorsal projections are not sufficient for sustenance of activity rhythms in constant darkness and this is suggestive of an additional component that is possibly dependent on sLNv molecular clock and PDF in sLNv soma. Additionally, despite loss of PERIOD in sLNv, their activity rhythms entrain to light/dark cycles indicating that sLNv molecular clocks are not necessary for entrainment. Under constant light, these flies lack PDF from both soma and dorsal projections of sLNv, and when subjected to light/dark cycles, show morning and evening anticipation and accurately phased morning and evening peaks. Thus, under light/dark cycles, PDF in sLNv is not necessary for morning anticipation.

Ataxin2 functions via CrebA to mediate Huntingtin toxicity in circadian clock neurons

“…To discover genes important for mHtt effects on circadian rhythms, we performed an RNAi screen to look for modifier effects of mHtt induced arrhythmicity by expressing HttQ128 in PDF+ LNv using PdfGAL4 ( PdfGAL4/UAS-HttQ128 ) [43, 55, 56] (S1 Fig). By day 10, we observe a substantial reduction in PDF+ sLNv cell body numbers consistent with published data (S1 Table) [55, 57]. As our previous data suggest that the circadian clock modifies mHtt effects [56], we focused on clock-controlled genes in PDF+ LNv.…”

“…Molecular mechanisms discovered in fly HD models are conserved with those found in mammalian models, including mTor-induced autophagy [45], histone acetylation [46–48], SUMOylation/ubiquitination [49], and axonal transport [50, 51]. mHtt also strongly disrupts sleep and/or circadian behavioral rhythms [6–8, 52–55] as well as selective loss of PDF in sLNv circadian clock neuron cell bodies [7, 55]. Despite the conservation of disrupted circadian rhythms in HD and HD models, the molecular mechanisms by which mHtt impacts circadian behavior remain unclear.…”

Waveform Plasticity under Entrainment to 12-h T-cycles in Drosophila melanogaster: Behavior, Neuronal Network, and Evolution

“…While it is possible that the second PDF peak in our experiment (Figs. 4 and 5b) is driven by the shorter peak of PER oscillation in the s-LNvs, it is also possible that the provided LDLD regime desynchronized the nuclear PER oscillation and PDF oscillation in the dorsal projections, a phenomenon that appears to have occurred in at least 2 previous manipulations (Kula et al, 2006; Prakash et al, 2017). Yet another possibility is that light directly affects properties of the LNvs such that PDF levels increase during the second photophase (similar to hyperexcitability-induced constitutive PDF expression in Nitabach et al, 2006), but in our case, PER oscillations are not acutely affected.…”

“…These flies were then sampled at 6 different phases (i.e., ZT03, ZT07, ZT11, ZT15, ZT19, and ZT23) while they were entrained to LDLD 5:7:5:7 for immunohistochemistry. The protocol we used is a slight modification of the method that has been published in a previous study (Prakash et al, 2017). We used control flies only to first gain insights into the behavior of the network under this novel short T -cycle regime.…”

“…Image Acquisition and Analysis of Intensities. All of the slides prepared during the immunohistochemistry experiment were imaged using confocal microscopy on a Zeiss LSM 880 microscope with a 40× (oil immersion) objective as described elsewhere (Prakash et al, 2017). We used a multiple component COSINORbased method (Cornelissen, 2014) to analyze aspects of rhythmicity in our intensity data, for both PER and PDF.…”