Is the novel rhythmically expressed gene 'quasimodo' the missing link between the circadian clock and membrane properties of pacemaker neurons?

Circadian clocks drive biological rhythms in many organisms including humans. They regulate our sleep/wake cycle, body temperature, and many other aspects of physiology and behaviour. Our master circadian clock resides in the brain, in a structure called the Supra Chiasmatic Nuclei (SCN). It consists of thousands of neurons which all express a set of so called 'clock genes'. These genes are special, since they regulate each other via feedback loops, meaning that the product of one gene represses another gene or even its own expression. As a result, molecular oscillations of many of these genes can be observed that occur with a frequency of ca. 24 hrs: our circadian molecular clock work! But this is not enough to achieve a circadianly rhythmic behaviour: For example to determine if we should be active or go to sleep, the SCN neurons somehow have to signal the time-of-day to other regions of our brain and body, for example to the pineal gland, which produces the 'sleeping hormon' melatonin when the SCN says so. SCN (or pacemaker) neurons do this, by producing electric signals (action potentials) in a rhythmic fashion; somehow regulated by the molecular clock. But how does this work? Recent evidence indicates that the properties of the neuronal membrane are rhythmically changed by the molecular clock, but again, the question remains how this is accomplished. In our model organism, the fruit fly Drosophila melanogaster, the situation is quite similar. It also has pacemaker neurons in its brain which express many clock genes in a circadian fashion. We recently isolated a novel rhythmically expressed gene called quasimodo (qsm), which is present in some of the clock neurons and is probably attached to the outside of their membranes. When we experimentally change the expression level of qsm (make more or less of it compared to normal flies), we induce very similar effects as if we manipulate genes that alter the electrical activity of neurons (called ion-channels). Therefore we believe that qsm interacts with at least some of these genes, and because its expression is controlled by the molecular clock, qsm could well be a factor that connects the clock with the neuronal membrane! To find this out we want to identify, which of the ion channel proteins interact with the Qsm protein and what exactly happens when we influence their expression. We also noticed that under certain circumstances of manipulating neuronal membrane proteins, we can observe strong behavioural rhythms in the absence of clock genes, which always were thought to be absolutely required for such rhythms. This is very astonishing and implies that there are rhythm-generating properties intrinsic to the neuronal membrane. We will try to find out what generates these rhythms by performing a mutagenesis screen where we will look for mutants that abolish the rhythms in the abnormally rhythmic flies. Our idea is that there are two rhythm-generating mechanisms: The known one consisting of the clock genes and their products, and another one, operating in the neuronal membranes. We think they are connected to each other, and with the experiments outlined in this proposal we want to show how the clock genes control the membrane oscillator and how in turn the membrane oscillator feeds back to control the clock genes.

Behavioural circadian rhythms in both mammals and flies are driven by specialized pacemaker neurons in the brain. Clock gene transcriptional feedback loops operate within these neurons, and regulate their rhythmic output. One such output is thought to be rhythmic electric activity, which could trigger rhythmic neuropeptide release thereby regulating behavioural rhythms. Electrical properties of the neuronal membrane are also crucial to maintain molecular rhythms in the nucleus. The experiments outlined here address how electrical activities can be regulated and controlled by the molecular feedback loops. We propose that the novel rhythmically expressed gene quasimodo (qsm), which encodes a putative membrane bound ZP-domain protein, is involved in this process. Our preliminary data show that over-expressing, or down-regulating qsm expression has similar effects on behavioural rhythms and neuropeptide accumulation as observed after manipulation of various ion channel genes. We identified a potential clock gene-independent and membrane-based circadian oscillator after interfering with qsm function. We plan to isolate proteins, which interact with the Qsm protein by various molecular and genetic approaches. Manipulating their function individually and in combination with altering qsm levels, will reveal if and how they are involved in circadian rhythm generation. By applying a Drosphila cell culture system we will determine the effects of Qsm and its interacting partners on the cellular distribution and release of the circadian neuropeptide PDF. Making use of our well established real-time luciferase reporter gene assay, we will investigate if the various membrane proteins feed back to the molecular clock by influencing clock gene expression. This will be the first comprehensive analysis aimed at isolating factors that regulate rhythmic events in circadian neuronal pacemaker membranes and their relation to the known rhythm entities.