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  • The first mammalian mutation linked


    The first mammalian mutation linked to CR was identified in golden hamsters (Mesocricetus auratus). The mutation was an autosomal allele first described in the late 1980s [8] and was identified using positional cloning [9]. These animals displayed a shortened period length (20h in homozygous animals) and were thus designated as τ mutants. Cloning revealed that the τ mutation was in casein kinase (CK) 1ε and decreased the maximal phosphorylation rates of CK1ε. There are several genes and their respective proteins that are involved with CR and a brief description of them follows. Cryptochrome (Cry) is a flavoprotein that acts as a blue light photoreceptor, directly modulating photo-input into the circadian clock. It has been shown that Cry associates with Per proteins [7] and is homologous to the protein Timeless (tim), first identified from Drosophila[1]. A mouse mutation generated through chemical mutagenesis and then screened for mutations, which alter CR, was mapped and the locus cloned using positional cloning [10], [11]. This protein was called Clock (Circadian Locomotor Output Cycles Kaput) and shown to be required for normal functioning of the circadian timing system. The Clock mutation in mice is a GSK2636771 of exon 19 and encodes a shorter protein [11], [12]. Clock interacts with a protein called Bmal1 (Brain and Muscle Aryl hydrocarbon receptor nuclear translocator ARNT-Like 1), which contains a basic helix–loop–helix protein domain. Bmal1 interacts with the Clock protein to form a heterodimer which activates transcription from genes containing an E-box in their promoters [7]. Periodicity in the SCN is generated by cyclic transcriptional/translational positive and negative feedback-loops of which Per, Cry, Clock, Bmal1, and their respective proteins are key components. The process by which light synchronizes (entrains) SCN periodicity to light/dark cycles is summarized as follows (Figure 1). Light signals, transmitted by the retinohypothalamic tract, initiate the transcription of Per by glutamate mediated activation of calcium channels and pituitary adenylate cyclase-activating polypeptide (PACAP) activation of the vasoactive intestinal peptide receptor 1 (VIPR-1) in SCN neurons [13]. VIPR-1 activation, and Ca influx through L channels, initiates phosphorylation of CREB, which subsequently binds to a CRE in the Per gene inducing its transcription [14]. As a result, cytoplasmic Per (protein) levels accumulate during the day and heterodimerize in the cytoplasm with the protein Cry. The Per-Cry complex translocates into the nucleus and inhibits the expression of Clock and Bmal1. The Clock and Bmal1 proteins heterodimerize to form a complex, which activates the transcription of genes containing an E-box structure, including the genes for Per and Cry. As Per levels increase throughout the day in the cytoplasm, the protein is phosphorylated by CK1ε or CK1δ and targeted for degradation by the ubiquitin ligase system [1], [15]. Blockade of CK1 prevents Per degradation and increases Per levels in the cytoplasm and the nucleus. It is this series of transcriptional, translational, and posttranslational activities that sets the timing of the endogenous clock. A secondary loop exists and functions to stabilize the core loop [15]. The stabilizing loop is driven by expression of the nuclear receptors Rev-erb α (also known as NR1D1—Nuclear Receptor subfamily 1, group D, member 1) and RORβ (RAR-related orphan receptor β, also known as NR1F2) [15], [16]. Rev-erb α functions as a negative regulator of Bmal1. Bmal1 interacts with Clock to drive expression of a set of genes with a common E-box promoter. One of these proteins is RORβ, which positively affects transcription of Bmal1, completing the stabilizing loop [15], [16]. Stabilization of the core loop is thought to preserve synchronized SCN function over time periods longer than a day [15], [16]. By inhibiting CK1, phosphorylation of Per could be blocked and this should alter the timing of the circadian clock, thus changing the rhythm of biological processes.