You just heard about a collection of conditions and illnesses that are downstream of the circadian clock. They're somehow a consequence of living against the clock, as what happens in shift work or social jet lag. There are also a set of illnesses that are ASSOCIATED with or CORRELATE with a malfunctioning clock. In the latter case, it's a hen and egg problem: Is it the clock that's causing the problem, or is the illness impacting the circadian clock? In the next two sections of the lecture, I want to touch on some of the examples where it's been experimentally demonstrated that clock genes contribute to altered and unhealthy behaviors. Or where they contribute to pathologies or unhealthy physiologies. What does it take to call the timing of sleep pathological or worthy of being called a "syndrome"? One of my favorite examples is Ludwig the IInd. He was the king of Bavaria, a large state in the South of Germany where this university is located, in the mid 1800´s. He was famous, for instance, for building the castle "Schloss Neuschwanstein". He was actually forcibly removed from office -he was diagnosed with schizophrenia without ever having been examined by the panel of doctors. If you're going to descriptions of Ludwig's lifestyle, you discover that, when he was allowed to control his environment, he arranged it so that he was awake at night and asleep during the day. He ordered breakfast at 6 in the evening, lunch at midnight, and dinner in the early morning hours. Now that's what I would call a problem chronotype. But mainly because -as the leader of a nation state- or if he'd been a student or a mother, or a father- he had responsibilities during the daytime. He would have surely been compromised in his performance if his circadian clock wasn't trained to the opposite time of day compared to that of those around him. An example of his poor performance might be his failed negotiations with Bismarck. Who essentially won the independence of Bavaria from Ludwig. As they say the rest is history. We'll never know if his clock genes were carrying an unusual mutation -he had no children- but the first subjects that eventually led to the description of the human circadian clock had extremely early sleep timing. Not quite as early as Ludwig but extreme nonetheless. We discussed this in the lecture on Molecular mechanisms. The geneticist that worked on these pedigrees found mutations in the Period-2 and Casein Kinase 1 genes. How does such a mutation lead to the extremely early chronotype in the affected individuals? This was worked out for the Per-2 mutation, which falls at the 662nd amino acid in the protein, a serine that's phosphorylated as part of the daily program. The mutation changes the amino acid from a serine to a glycine. The consequence of this change is that this position in the protein can no longer be phosphorylated. Recall that the phosphorylation state specifies many things, including setting proteins up for degradation, changing their sub cellular localization, and potentiating them as transcription factors. In the case of PER2 the mutation found in the family with early sleep timing showed a more rapid degradation of the protein. In this experiment about half of the protein remains after two hours when the mutation is present. In the wild type protein, half is still present after four hours. A similar observation has been reported for the FREQUENCY protein in Neurospora, suggesting that this may be a common theme in clock regulation. Interestingly, a second effect of the PER2 mutation is seen in its sub cellular localization. The mutant PER2 sequence is cleared from the nucleus, more rapidly than the wild type PER2. Obviously, the protein can't function as a transcription factor if it's not in the nucleus. An additional study found that indeed also the transcription factor potential of the molecule was altered. So with a single mutation three distinct phosphorylation mediated processes are potentially altered. One idea for how this works is that certain phosphorylation sites act as a sort of a gateway to additional phosphorylation. So it could be a certain area that's mediating the degradation defect, and another area mediating the sub cellular localization with both of them being dependent on an upstream phosphorylation event at the mutant site. If we find humans with extreme chronotypes that furthermore have mutations in clock genes, then we might also expect to find some animals with this phenotype if they are mutant in their clock genes. Alternatively, the timing of sleep of mutants made in the lab, random mutations produced in mutant screens, these might show ultra sleep behavior, revealing new clock genes true on both counts. Actually, the first animal mutant was the rather random appearance of a hamster. That became active far too early in a light dark cycle. You may recall that the nocturnal rodents are active only in the dark phase, like you see here, with a small amount of activity starting to come up in the late part of the light phase. Still mostly masked, or suppressed, by the light. The mutant animals would start being active well before the lights went out. These are a bit like the Advance sleep phase syndrome families. In constant conditions, the homozygous animals showed an extremely short free running period of about 20 hours. The correlation here between period -very short- and entrained phase -very early- contributed to the concept of the period phase relationship. You might note that while there are many examples where this relationship holds as you've heard in the entrainment lecture there are also many exceptions. The mutation in this animal was found in the Casein kinase 1 gene, by the way. A number of mutants have been identified through mutant screens: Mutant mice are produced and then tested for a number of behaviors, including the timing of the activity and quiescence. The after hours mutant shows very late activity onset in a light/dark cycle. When this mutation was located, it was found in an Fbox protein, a ubiquitin E3 ligase. These proteins are dedicated actors in the protein degradation process and their mutation leads to abnormal stability of -at least- clock proteins. They are also part of the clock in plants, fungi, and flies. Another molecular clocks principle. The take home message here is that changes in the sequence of clock proteins can lead to substantial changes in the timing of sleep. That this is labeled a syndrome reflects the substantial disruption on social life for those who are living at different times than the rest of us. [SOUND]
