Brigitte Grima

Morning and evening peaks of activity rely on different clock neurons of the Drosophila brain

In Drosophila, a ‘clock’ situated in the brain controls circadian rhythms of locomotor activity. This clock relies on several groups of neurons that express the Period (PER) protein, including the ventral lateral neurons (LNvs), which express the Pigment-dispersing factor (PDF) neuropeptide, and the PDF-negative dorsal lateral neurons (LNds). In normal cycles of day and night, adult flies exhibit morning and evening peaks of activity; however, the contribution of the different clock neurons to the rest–activity pattern remains unknown. Here, we have used targeted expression of PER to restore the clock function of specific subsets of lateral neurons in arrhythmic per0 mutant flies. We show that PER expression restricted to the LNvs only restores the morning activity, whereas expression of PER in both the LNvs and LNds also restores the evening activity. This provides the first neuronal bases for ‘morning’ and ‘evening’ oscillators in the Drosophila brain. Furthermore, we show that the LNvs alone can generate 24 h activity rhythms in constant darkness, indicating that the morning oscillator is sufficient to drive the circadian system.

The F-box protein slimb controls the levels of clock proteins period and timeless.

The Drosophila circadian clock is driven by daily fluctuations of the proteins Period and Timeless, which associate in a complex and negatively regulate the transcription of their own genes. Protein phosphorylation has a central role in this feedback loop, by controlling Per stability in both cytoplasmic and nuclear compartments as well as Per/Tim nuclear transfer. However, the pathways regulating degradation of phosphorylated Per and Tim are unknown. Here we show that the product of the slimb (slmb) gene—a member of the F-box/WD40 protein family of the ubiquitin ligase SCF complex that targets phosphorylated proteins for degradation—is an essential component of the Drosophila circadian clock. slmb mutants are behaviourally arrhythmic, and can be rescued by targeted expression of Slmb in the clock neurons. In constant darkness, highly phosphorylated forms of the Per and Tim proteins are constitutively present in the mutants, indicating that the control of their cyclic degradation is impaired. Because levels of Per and Tim oscillate in slmb mutants maintained in light:dark conditions, light- and clock-controlled degradation of Per and Tim do not rely on the same mechanisms.

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.

Analysis of the 5' region of the human tyrosine hydroxylase gene: combinatorial patterns of exon splicing generate multiple regulated tyrosine hydroxylase isoforms

A single human gene has been described to encode multiple tyrosine hydroxylase (TH) mRNAs. The study of this variation has been extended by S1 mapping experiments and by analysis of the 5’region of the TH gene. Four different mRNAs were found to originate solely from alternative splicing of two exons. Comparison of the 5’flanking regions of human and rat genes discloses several highly conserved segments, likely to play an important role in the regulation of TH gene expression.

A New Family of Transcripts of the Myelin Basic Protein Gene: Expression in Brain and in Immune System

Abstract: A cDNA clone (MBP2) corresponding to a novel mouse myelin basic protein (MBP) mRNA has been isolated from an adult mouse bone marrow cDNA library. It contains the MBP exons la‐7 except exon 5. Using PCR experiments we have determined that this MBP2 mRNA belongs to a new MBP mRNA family initiated upstream from exon 1b. Their 5’end extends into exon la and/or the region O’previously described. These mRNAs are generated by alternative splicing of the primary transcript involving excision of exon la, 1b, 2, 5, or 6. Thus, these new mRNAs are produced from a promoter(s) located upstream from the major promoter 1b. They are expressed in brain (at least from embryonic day 15), in bone marrow, and in other hemolymphoppietic tissues, particularly in macrophage cells. As their expression is not restricted to myelinating cells, the function of these novel MBP mRNAs and putative proteins might not be related to myelination.

A Novel Transcript Overlapping the Myelin Basic Protein Gene

Abstract: Myelin basic protein (MBP) is a major constituent of myelin synthesized by oligodendrocytes and Schwann cells. We have investigated the expression of mouse MBP RNAs outside the nervous system. Nuclease protection experiments indicate that RNAs containing exon 1 and not the six downstream exons of the MBP gene are transcribed in various hemopoietic tissues. We have isolated a hemopoietic MBP‐related (HMBPR) cDNA clone from a mouse bone marrow cDNA library screened with an MBP cDNA probe. This clone contains exons 1a and 1b and a part of intron 1 of the MBP gene. An additional 5’region, encoded by at least three unidentified exons, lies upstream of exon 1a. The HMBPR clone corresponds to a 5‐kb RNA expressed in bone marrow, spleen, thymus, and macrophagic cells. This transcript is expressed at a similar level in brain, although at a lower level than the classical 2‐kb mRNA. These data indicate that a new transcript, overlapping the MBP transcription unit and controlled by a distinct promoter, is expressed in hemopoietic tissues. This RNA might encode a 21‐kDa protein sharing a common domain with MBP.

Cloning of Quail Tyrosine Hydroxylase: Amino Acid Homology with Other Hydroxylases Discloses Functional Domains

Abstract: A cDNA clone containing the entire coding region of quail tyrosine hydroxylase (TH) has been isolated and analyzed. Comparison with rat and human THs and phenylalanine hydroxylases reveals several highly conserved domains. Two of them, shared by all these hydroxylases, are localized in the central and C‐terminal parts of the molecules, and most probably include the active site. Two others are found only in the TH molecules. One contains putative sites of phosphorylation and is implicated in the posttranslational regulation of the enzyme. The second highly preserved domain, consisting of a stretch of 21 amino acids, is presumably associated with an important feature of the enzyme that remains to be identified.

Isolation of a rat pineal gland cDNA clone homologous to tyrosine and phenylalanine hydroxylases

A rat pineal gland cDNA expression library has been probed with an antiserum raised against rat tryptophan hydroxylase. A clone has been isolated and its sequence reveals a high degree of homology with those of tyrosine and phenylalanine hydroxylases.

CULLIN-3 Controls TIMELESS Oscillations in the Drosophila Circadian Clock

Eukaryotic circadian clocks rely on transcriptional feedback loops. In Drosophila, the PERIOD (PER) and TIMELESS (TIM) proteins accumulate during the night, inhibit the activity of the CLOCK (CLK)/CYCLE (CYC) transcriptional complex, and are degraded in the early morning. The control of PER and TIM oscillations largely depends on post-translational mechanisms. They involve both light-dependent and light-independent pathways that rely on the phosphorylation, ubiquitination, and proteasomal degradation of the clock proteins. SLMB, which is part of a CULLIN-1-based E3 ubiquitin ligase complex, is required for the circadian degradation of phosphorylated PER. We show here that CULLIN-3 (CUL-3) is required for the circadian control of PER and TIM oscillations. Expression of either Cul-3 RNAi or dominant negative forms of CUL-3 in the clock neurons alters locomotor behavior and dampens PER and TIM oscillations in light-dark cycles. In constant conditions, CUL-3 deregulation induces behavioral arrhythmicity and rapidly abolishes TIM cycling, with slower effects on PER. CUL-3 affects TIM accumulation more strongly in the absence of PER and forms protein complexes with hypo-phosphorylated TIM. In contrast, SLMB affects TIM more strongly in the presence of PER and preferentially associates with phosphorylated TIM. CUL-3 and SLMB show additive effects on TIM and PER, suggesting different roles for the two ubiquitination complexes on PER and TIM cycling. This work thus shows that CUL-3 is a new component of the Drosophila clock, which plays an important role in the control of TIM oscillations.