André Klarsfeld

Defining the role of Drosophila lateral neurons in the control of circadian rhythms in motor activity and eclosion by targeted genetic ablation and PERIOD protein overexpression.

The ventral lateral neurons (LNvs) of the Drosophila brain that express the period (per) and pigment dispersing factor (pdf) genes play a major role in the control of circadian activity rhythms. A new P‐gal4 enhancer trap line is described that is mostly expressed in the LNvs This P‐gal4 line was used to ablate the LNvs by using the pro‐apoptosis gene bax, to stop PER protein oscillations by overexpressing per and to block synaptic transmission with the tetanus toxin light chain (TeTxLC). Genetic ablation of these clock cells leads to the loss of robust 24‐h activity rhythms and reveals a phase advance in light–dark conditions as well as a weak short‐period rhythm in constant darkness. This behavioural phenotype is similar to that described for disconnected1 (disco1) mutants, in which we show that the majority of the individuals have a reduced number of dorsally projecting lateral neurons which, however, fail to express PER. In both LNv‐ablated and disco1 flies, PER cycles in the so‐called dorsal neurons (DNs) of the superior protocerebrum, suggesting that the weak short‐period rhythm could stem from these PDF‐negative cells. The overexpression of per in LNs suppresses PER protein oscillations and leads to the disruption of both activity and eclosion rhythms, indicating that PER cycling in these cells is required for both of these rhythmic behaviours. Interestingly, flies overexpressing PER in the LNs do not show any weak short‐period rhythms, although PER cycles in at least a fraction of the DNs, suggesting a dominant role of the LNs on the behavioural rhythms. Expression of TeTxLC in the LNvs does not impair activity rhythms, which indicates that the PDF‐expressing neurons do not use synaptobrevin‐dependent transmission to control these rhythms.

Novel Features of Cryptochrome-Mediated Photoreception in the Brain Circadian Clock of Drosophila

In Drosophila, light affects circadian behavioral rhythms via at least two distinct mechanisms. One of them relies on the visual phototransduction cascade. The other involves a presumptive photopigment, cryptochrome (cry), expressed in lateral brain neurons that control behavioral rhythms. We show here that cry is expressed in most, if not all, larval and adult neuronal groups expressing the PERIOD (PER) protein, with the notable exception of larval dorsal neurons (DN2s) in which PER cycles in antiphase to all other known cells. Forcing cry expression in the larval DN2s gave them a normal phase of PER cycling, indicating that their unique antiphase rhythm is related to their lack of cry expression. We were able to directly monitor CRY protein in Drosophila brains in situ. It appeared highly unstable in the light, whereas in the dark, it accumulated in both the nucleus and the cytoplasm, including some neuritic projections. We also show that dorsal PER-expressing brain neurons, the adult DN1s, are the only brain neurons to coexpress the CRY protein and the photoreceptor differentiation factor GLASS. Studies of various visual system mutants and their combination with the cryb mutation indicated that the adult DN1s contribute significantly to the light sensitivity of the clock controlling activity rhythms, and that this contribution depends on CRY. Moreover, all CRY-independent light inputs into this central behavioral clock were found to require the visual system. Finally, we show that the photoreceptive DN1 neurons do not behave as autonomous oscillators, because their PER oscillations in constant darkness rapidly damp out in the absence of pigment-dispersing-factor signaling from the ventral lateral neurons.

Five Listeria monocytogenes genes preferentially expressed in infected mammalian cells: plcA, purH, purD, pyrE and an arginine ABC transporter gene, arpJ

Listeria monocytogenes is a bacterial pathogen that multiplies within the cytosol of eukaryotic cells. To identify Listeria genes with preferentially intracellular expression (pic genes), a library of Tn917‐lac Insertion mutants was screened for transcriptional fusions to lacZ with higher expression inside a macrophage‐like cell line than in a rich broth medium. Five pic genes with up to 100‐fold induction inside cells were identified. Three of them (purH, purD and pyrE) were involved in nucleotide biosynthesis. One was part of an operon encoding an ABC (ATP‐binding cassette) transporter for arginine. The corresponding mutants were not affected in intracellular growth, cell‐to‐cell spread or virulence, except for the transporter mutant, whose LD50 after intravenous infection of mice was twofold higher than the wild‐type. The fifth gene was plcA, a previously identified virulence gene that encodes a phosphatidylinositol‐phospholipase C., and is cotranscribed with prfA, a gene encoding a pleiotropic transcriptional activator of known virulence genes. Although plcA expression is known to depend on PrfA, a prfA promoter–lacZ fusion was highly expressed both Inside and outside cells. Furthermore, in the presence of cellobiose, a disac‐charide recently shown to repress plcA and hly expression, plcA and hly mRNA levels were dramatically reduced without any decrease in the monocistronic prfA mRNA levels. These results demonstrate that virulence gene activation does not depend only on prfA transcript accumulation.

Effects of circadian mutations and LD periodicity on the life span of Drosophila melanogaster.

The pervasive occurrence of circadian clocks throughout the living world underlines their adaptive value. Nonetheless, there is surprisingly little evidence for a negative impact, on any animal species, of a constant discrepancy between the environmental and endogenous periods. Male Drosophila melanogaster per mutants with altered circadian periods were compared to the wild type in two different LD schedules. Life span was used as a global index of physiological adaptation. The life span of the mutants was significantly reduced by up to 15% for the flies whose period differs most from that of the wild type. A reduction was observed even when flies were kept in an LD schedule fitting a mutant period. The LD schedule made no significant difference on its own, but the authors found evidence for an interaction between genotype and LD schedule in determining life span. These results are consistent with the importance of the circadian clock in maintaining internal temporal order independent of environ-mental cycles. Nonetheless, a large difference between the environmental and endogenous periods has a measurable impact.

Circadian rhythms of locomotor activity in Drosophila

Drosophila is by far the most advanced model to understand the complex biochemical interactions upon which circadian clocks rely. Most of the genes that have been characterized so far were isolated through genetic screens using the locomotor activity rhythms of the adults as a circadian output. In addition, new techniques are available to deregulate gene expression in specific cells, allowing to analyze the growing number of developmental genes that also play a role as clock genes. However, one of the major challenges in circadian biology remains to properly interpret complex behavioral data and use them to fuel molecular models. This review tries to describe the problems that clockwatchers have to face when using Drosophila activity rhythms to understand the multiple facets of circadian function.

PDF-modulated visual inputs and cryptochrome define diurnal behavior in Drosophila

Morning and evening circadian oscillators control the bimodal activity of Drosophila in light-dark cycles. The lateral neurons evening oscillator (LN-EO) is important for promoting diurnal activity at dusk. We found that the LN-EO autonomously synchronized to light-dark cycles through either the cryptochrome (CRY) that it expressed or the visual system. In conditions in which CRY was not activated, flies depleted for pigment-dispersing factor (PDF) or its receptor lost the evening activity and displayed reversed PER oscillations in the LN-EO. Rescue experiments indicated that normal PER cycling and the presence of evening activity relied on PDF secretion from the large ventral lateral neurons and PDF receptor function in the LN-EO. The LN-EO thus integrates light inputs and PDF signaling to control Drosophila diurnal behavior, revealing a new clock-independent function for PDF.

A Role for Blind DN2 Clock Neurons in Temperature Entrainment of the Drosophila Larval Brain

Circadian clocks synchronize to the solar day by sensing the diurnal changes in light and temperature. In adult Drosophila, the brain clock that controls rest–activity rhythms relies on neurons showing Period oscillations. Nine of these neurons are present in each larval brain hemisphere. They can receive light inputs through Cryptochrome (CRY) and the visual system, but temperature input pathways are unknown. Here, we investigate how the larval clock network responds to light and temperature. We focused on the CRY-negative dorsal neurons (DN2s), in which light–dark (LD) cycles set molecular oscillations almost in antiphase to all other clock neurons. We first showed that the phasing of the DN2s in LD depends on the pigment-dispersing factor (PDF) neuropeptide in four lateral neurons (LNs), and on the PDF receptor in the DN2s. In the absence of PDF signaling, these cells appear blind, but still synchronize to temperature cycles. Period oscillations in the DN2s were stronger in thermocycles than in LD, but with a very similar phase. Conversely, the oscillations of LNs were weaker in thermocycles than in LD, and were phase-shifted in synchrony with the DN2s, whereas the phase of the three other clock neurons was advanced by a few hours. In the absence of any other functional clock neurons, the PDF-positive LNs were entrained by LD cycles but not by temperature cycles. Our results show that the larval clock neurons respond very differently to light and temperature, and strongly suggest that the CRY-negative DN2s play a prominent role in the temperature entrainment of the network.

Circadian Synchronization and Rhythmicity in Larval Photoperception-Defective Mutants of Drosophila

A single light episode during the first larval stage can set the phase of adult Drosophila activity rhythms, showing that a light-sensitive circadian clock is functional in larvae and is capable of keeping time throughout development. These behavioral data are supported by the finding that neurons expressing clock proteins already exist in the larval brain and appear to be connected to the larval visual system. To define the photoreceptive pathways of the larval clock, the authors investigated circadian synchronization during larval stages in various visual systems and/or cryptochrome-defective strains. They show that adult activity rhythms cannot be entrained by light applied to larvae lacking both cryptochrome and the visual system, although such rhythms were entrained by larval stage-restricted temperature cycles. Larvae lacking either pathway alone were light entrainable, but the phase of the resulting adult rhythm was advanced relative to wild-type flies. Unexpectedly, adult behavioral rhythms of the glass 60 j and norpA P 24 visual system mutants that were entrained in the same conditions were found to be severely impaired, in contrast to those of the wild type. Extension of the entrainment until the adult stage restored close to wild-type behavioral rhythms in the mutants. The results show that both cryptochrome and the larval visual system participate to circadian photoreception in larvae and that mutations affecting the visual system can impair behavioral rhythmicity.

Human cryptochrome-1 confers light independent biological activity in transgenic drosophila correlated with flavin radical stability

Cryptochromes are conserved flavoprotein receptors found throughout the biological kingdom with diversified roles in plant development and entrainment of the circadian clock in animals. Light perception is proposed to occur through flavin radical formation that correlates with biological activity in vivo in both plants and Drosophila. By contrast, mammalian (Type II) cryptochromes regulate the circadian clock independently of light, raising the fundamental question of whether mammalian cryptochromes have evolved entirely distinct signaling mechanisms. Here we show by developmental and transcriptome analysis that Homo sapiens cryptochrome - 1 (HsCRY1) confers biological activity in transgenic expressing Drosophila in darkness, that can in some cases be further stimulated by light. In contrast to all other cryptochromes, purified recombinant HsCRY1 protein was stably isolated in the anionic radical flavin state, containing only a small proportion of oxidized flavin which could be reduced by illumination. We conclude that animal Type I and Type II cryptochromes may both have signaling mechanisms involving formation of a flavin radical signaling state, and that light independent activity of Type II cryptochromes is a consequence of dark accumulation of this redox form in vivo rather than of a fundamental difference in signaling mechanism.

Drosophila Clock Is Required in Brain Pacemaker Neurons to Prevent Premature Locomotor Aging Independently of Its Circadian Function

Circadian clocks control many self-sustained rhythms in physiology and behavior with approximately 24-hour periodicity. In many organisms, oxidative stress and aging negatively impact the circadian system and sleep. Conversely, loss of the clock decreases resistance to oxidative stress, and may reduce lifespan and speed up brain aging and neurodegeneration. Here we examined the effects of clock disruptions on locomotor aging and longevity in Drosophila. We found that lifespan was similarly reduced in three arrhythmic mutants (ClkAR, cyc0 and tim0) and in wild-type flies under constant light, which stops the clock. In contrast, ClkAR mutants showed significantly faster age-related locomotor deficits (as monitored by startle-induced climbing) than cyc0 and tim0, or than control flies under constant light. Reactive oxygen species accumulated more with age in ClkAR mutant brains, but this did not appear to contribute to the accelerated locomotor decline of the mutant. Clk, but not Cyc, inactivation by RNA interference in the pigment-dispersing factor (PDF)-expressing central pacemaker neurons led to similar loss of climbing performance as ClkAR. Conversely, restoring Clk function in these cells was sufficient to rescue the ClkAR locomotor phenotype, independently of behavioral rhythmicity. Accelerated locomotor decline of the ClkAR mutant required expression of the PDF receptor and correlated to an apparent loss of dopaminergic neurons in the posterior protocerebral lateral 1 (PPL1) clusters. This neuronal loss was rescued when the ClkAR mutation was placed in an apoptosis-deficient background. Impairing dopamine synthesis in a single pair of PPL1 neurons that innervate the mushroom bodies accelerated locomotor decline in otherwise wild-type flies. Our results therefore reveal a novel circadian-independent requirement for Clk in brain circadian neurons to maintain a subset of dopaminergic cells and avoid premature locomotor aging in Drosophila.