di
Anna Giulia Cattaneo, M.D.

DBSM - University of Insubria, Via J-H Dunant 3 - 21000 Varese.

Changes in sleep quality and rhythms are frequent complaints in elder subjects: diurnal sleepiness, nocturnal periods of wakefulness and early awakenings became more frequent as aging progresses, and cause enhanced intake of hypnotic medications. Because of sleep-wake rhythm shows a circadian pattern, altered circadian clock regulation in advanced age has been advocated as a possible causative mechanism explaining insomnia in this range of age.
In spite of several clinical findings partially supporting this hypothesis, the problem seems to be more complex and involving at least two different pathways, evolved in Vertebrates and in Mammals.

While in lower animals circadian clock and biochemical changes linked to the environmental light are linked, in Vertebrates and in Mammals two distinct functions have been developed, one mediated by molecules mainly expressed in the retina and influenced by the light in the environment, the other showing an intrinsic cyclic activity. These last can be defined as mechanism whit a near 24 hours-periodicity, determined by the activity of several regulatory genes which are located in the bilaterally paired suprachiasmatic nuclei (SCN) of the hypothalamus. Their expression shows oscillations within 24 hours, persistent even in the absence of environmental stimuli like light.

Both these mechanisms have been advocated for explaining sleep disruption and deprivation.

A summary of the main features of these genes in humans beings and of their interactions will precede the description of some methods used to study the circadian clock in humans and results found in aging subjects.

Main circadian cycle regulators in humans and Mammals CNS.

1. Gene Clock. Hortolog of the gene found in mouse, its transcript is a Hypoxia Inducible Factor (HIF), belonging to the basic helix-loop-helix (bHLH) family of transcription factors and having a Hat (histone acetyltransferase). Chromosome: 4, 4; Locus: 4q12. Genetic polymorphism has been related to mental illness (schizophrenia and bipolarism) and possibly to sleep disorders.
2. ARNTL (Bmal1). Its transcript, a basic helix-loop-helix protein, dimerizes with the transcript of the CLOCK gene, enhancing the HAT activity. chromosome: 11; Location: 11p15
3. PER. As recalled before, this gene is a hortolog of the PERIOD (PER) of Drosophila (44% homology) and two point mutations in the transcript are responsible for two types of a rare human inherited disease known as familial advanced sleep phase syndrome (FASPS). Even in Drosophila the activity of this gene is related to the sleep timing... In humans the gene encodes a preprotein containing a basic helix-loop-helix (bHLH) that gives rise for
4. postranscriptional splicing to three 3 transcripts of 4.7, 3.0, and 6.6 kb: only two of them seems to be regulators of circadian oscillations PER3 being expressed outside the SNC and apparently deprived of oscillatory properties. Activity: histone acetylation and deacetylation. Chromosome: 17; Locus: 17p13.1-p12
5. CRY1.The gene encodes for the cryptochrome, a blue-light photoreceptor, similar to photolyase found in plants. Photolyases contain flavin adenine dinucleotide (FAD), and a second, variable chromophore. They mediate photo reactivation, a repair mechanism that removes UV-induced DNA damage. Class I photolyases include microbial photolyases and several plant blue-light photoreceptors. The gene found in humans (and Mammals) is expressed in the brain, and despite the dependence from the light of several functions, shows a cyclic activity not dependent from the light cycle, so belonging to the group of circadian regulators as defined before. The transcript is located in the mitochondrion. Chromosome: 12; Location: 12q23-q24.1.

6. MTNR1A. Melatonin is one of at least two recognized markers of circadian rhythmicity in Mammals. This gene encodes one of two melatonin receptor, a G-protein integral to the membrane of neurons in the hypothalamic suprachiasmatic nucleus, where it should be be involved in circadian rhythm, and in the hypophysial pars tuberalis, where it mediates the reproductive effects of melatonin.

Chain of events:

1. Clock and Bmal gene activation
2. Heterodimerization CLOCK/Bmal
3. Drives of reported gene PER (histone acetylation and deacetylation)
4. CRY1 and CRY activation (delayed)
5. PER and CRY proteins autorepression.
6. CRY in turn represses CLOCK/BMAL1 transcriptional activity by an unknown mechanism.
7. MTNR1B product inhibits circadian firing rhythms in the SCN.

Molecules expressed in the retina having a light-related cycling

1. CRY2. The transcript of this gene is similar to the class II photolyases which have been found in higher eukaryotes. It is highly expressed in the retina, and has been described as a vitamin A-based opsin, a blue-light photoreceptor necessary for vision.
2. MTNR1B. Like MTNR1A, the product of this gene is a transmembrane G protein able to bind melatonin. However the gene is mainly expressed in the retina, and minimally in the CNS, suggesting its action being involved in cycling linked to daylight or environmental light.
3. PER3. This transcript of gene PERIOD, in contrast with PER1 and 2, does not show an intrinsic periodicity. Its cycling is linked to photoperiodicism and timing is similar to that of MTNRB1.

The activity of circadian rhythm regulators has been documented in rodents, in which the PER1 gene is mainly expressed during the nocturnal activity period, PER2 is synchronous with CRY1 and peaks early in the morning, while CLOCK peaks independently, late in the evening.

In humans, clinical investigations documenting the circadian rhythms are mainly based on the phases of melatonin and body temperature.

In aged people and advance of the circadian clock seems to be related with proportional advances in sleep time: the aged go to sleep and wake too early. Similar features have been described in a rare human inherited disease known as familial advanced sleep phase syndrome (FASPS) and affecting even young people, with major disturbance of sleep timing and sleep deprivation. The disease is supported by point mutations of one of the genes regulating the circadian clock, and namely PER2. However, the only finding of sleep time advance is not sufficient to explain the complexity of sleep disruption found in many older people, affecting the sleep architecture and the slow-wave and REM phases. Such kind of alterations are found in PER3 gene polymorphism, and in old subjects submitted to a higher amount of light during the nocturnal rest, as could be the case for patients lying in nursing home.
Under this point of view, it seems possible to conclude that the insomnia in older subjects can be only in part linked to the circadian clock advance that seems to be the main alteration characterizing this mechanism during the aging process. A role of peripheral retinal mechanisms whose activity is dependent from the light and dark cycle is possibly involved.

Several studies claim that better regulation of environmental light quality during the night, or light and dark cycles, can possibly be useful in limiting the disturbance of sleep in these individuals, at least in part. A reasonable amount of physical activity, as prudent for the clinical conditions of the aged patient, can also be of some help.
Pharmacological treatment with non traditional drugs, like chronobiotics, of the age-related insomnia, need enhanced knowledges to be considered safe and efficacious.

The applications of mathematical modelling to circadian cycles could open a new way to pharmacodynamic; with the restraint that severe scientific control will be applied.