The ninna nanna gene: an insight into an archetypal circuit integrating circadian and homeostatic drive to regulate sleep

Why we sleep remains an unresolved mystery of biology. Why do humans have to spend one-third of their lifetime in a status of profound unconsciousness which leaves them vulnerable and endangered? What do we gain from it? We still do not possess an answer to this question but we assume that it must be something tremendously important, also considered that sleep appears to be a necessity not just in humans but in all animals - including fruit flies. A particularly intriguing evolutionary conserved feature of sleep is what we call "sleep homeostasis", that is: the innate modulation of sleep pressure based on previous sleep amount. If we have a good long nap, we may have a harder time falling asleep at night; conversely, if we pull an all-nighter partying on Sunday night, we are going to have a hard time at the office on the following morning. That is sleep homeostasis.

In all animals, sleep is considered to be under control of two independent mechanisms: a homeostatic regulator and a circadian regulator. This so-called "two-process model" was formulated more than 30 years ago by Borbély and it is still universally considered the reference model for sleep regulation. However, after three decades from its formulation, the very biology underlying this model remains elusive and we still ignore how the homeostatic and circadian processes integrate at a cellular & biological level. How do the two intrinsic drives that command sleep pressure integrate at a cellular level? What are the neurons and genes involved with this process?

We recently characterised a new gene - called "ninna nanna" after the Italian term for lullaby - that appears to be involved with this process. The ninna nanna gene is expressed in the brain of fruitflies in a minute but functionally important neuronal circuit that appears to encompass neurons involved with the circadian clock and neurons involved with the perception of homeostatic pressure.

We do not know the molecular function of the gene but we know that encodes for two enzymes with a predicted affinity for slightly different molecules. We propose that these molecules (NADH and NADPH) could work as a molecular marker for sleep pressure, for instance increasing or decreasing in amount when we are more or less tired.

This is the first time that a neuronal circuit integrating the two processes underpinning sleep regulation has been sketched and we aim to study it in greater detail to ultimately understand the biology of sleep.

In all animals, sleep pressure is under continuous tight regulation. It is universally accepted that this regulation is described by a two-process model, integrating both a circadian and a homeostatic controller. From a biological perspective, much is known about the molecular and cellular underpinnings of the circadian regulator, but very little about the homeostatic regulator - and even less on how these two interact to create a functional "somnostat".
Here we present a Drosophila model of a somnostat, that - for the first time - could be used to study how circadian rhythms and homeostatic sleep pressure coordinate an animal's drive to sleep. We have identified a gene, ninna nanna, whose expression labels the two complementary components of a functional sleep-regulating circuit. Peculiarly, the ninna nanna gene gives rise to two alternatively spliced isoforms: one (ninna) is expressed and functions in the presynaptic, circadian component of the circuit; the second (nanna) is expressed and functions in the postsynaptic, homeostatic component of the circuit.

Our underlying working hypothesis is that ninna and nanna may act as molecular sensors for intracellular levels of NADP(H )and NAD(H), their respective co-factors.
In this project we will:
# visualise and interfere with the neuronal levels of NAD(P)(H) at the single cell level, targeting candidate regulatory neurons, using existing and novel genetic tools;
# dissect the anatomical and functional connectivity of the archetypal ninna nanna circuit, using modern tools of neuronal connectivity and optogenetics;
# explore a potential role for other genes in the same family of ninna nanna, using RNAi and CRISPR

Importantly, our laboratory is a leading laboratory in the field of Drosophila sleep. Not only do we possess all the expertise to carry the proposed experimental work, but we also created some of the most advanced, and now commonly used, software and hardware tools to study behaviour in flies.

This work can impact mainly at two levels:

1. Scientific impact
Providing a new framework for studying how circadian rhythms integrate with homeostatic drive would provide for the first time with a biological sandbox for the so-called "two-process model" of sleep regulation. Not just our Drosophila colleagues but the sleep field at large will benefit from having access to a genetically amenable model of neurons integrating circadian rhythms and homeostatic process. The Drosophila model for sleep is a recent one, having been officially introduced in 2000. Yet, with almost 600 publications, flies have already played a pivotal role in our understanding of sleep, uncovering new genes and highlighting new circuits. Because sleep is such a conserved phenomenon in the animal kingdom, every biological component that is found in flies is likely to reflect a general principle that will help to elucidate the problem in humans too. For biology, genetics, and behaviour, sleep has many parallels with the field of circadian rhythms. Drosophila has been instrumental to understand the former and there is no reason to think they will not be equally instrumental in understanding the latter. Finally, the fact that Drosophila could be used to study the regulation of sleep has a strong appeal in scientific terms but also in terms of animals replacement (3Rs).

2. Societal impact
Impact on society of study like this one could be very big. We would show the public that reductionists approaches are still very powerful in science and highlight, once more, the power of employing Drosophila even when studying subjects that are complex as sleep. We have a strong track record of bringing our research to the public, with frequent schools seminars and workshop; large public engagements; visibility on the internet and on social networks; TED talks (see https://lab.gilest.ro/outreach/ ). In our experience, the use of Drosophila to study sleep is always fantastically well received, for it merges two fascinating aspects of research that happen to be understandable and understood by the many.