Z49859

a The number represents position of the histidine residues.

a The number represents position of the histidine residues.

Because the tissue specificity and circadian expression of PINA correlated well with NAT expression and melatonin synthesis, we suspected that PINA was involved in melatonin biosynthesis. Our investigations of PINA/ATP7B mutant LEC rats indeed revealed a defect in NAT protein stability, but, unfortunately, this was caused by an independent mutation in the NAT gene and not by the PINA mutation. We have now generated a novel line of rats in which three of the mutant LEC genes encoding NAT, PINA, and coat color have now been segregated: LPP (PINA-), LPN (NAT-), and LPW (wild type in all three loci). Using these strains of rats, we were able to address the role of PINA in normal pineal physiology. Using a novel on-line pineal microdialysis protocol, we have measured the circadian production of melatonin and its precursors in animals quantitatively and reproducibly. In normal rats (LPW; Fig. 4), we see low melatonin levels during the day and a sharp rise in melatonin synthesis shortly after lights off; melatonin output dramatically drops off minutes before lights on. The melatonin synthetic profile of LPP animals shows no significant differences over the course of the day or the night (LPP, Fig. 4), demonstrating that PINA is not essential for melatonin synthesis and secretion. Thus, the natural role of PINA remains unresolved presently.

Other postulated functions of PINA must account for its tissue distribution and temporal expression pattern, which suggest involvement in a pineal/retinal-specific nocturnal process. Melatonin

1:00 11:00 1:00 11:00 time of the day time of the day

Fig. 4. Diurnal profile of pineal N-acetylserotonin (NAS, solid circle) and melatonin (open circle) release in wild-type (LPW) and PINA-defective (LPP) rats as determined by in vivo pineal microdialysis (14). Lights were off at 1 am and on at 11 am (within the shaded area). The diurnal pattern is reproducible in 100% of the animals examined, with slight variations in the level of melatonin among individual rats. There is no statistical difference between LPP and LPW rat strains in the level of pineal melatonin secretion.

1:00 11:00 1:00 11:00 time of the day time of the day

Fig. 4. Diurnal profile of pineal N-acetylserotonin (NAS, solid circle) and melatonin (open circle) release in wild-type (LPW) and PINA-defective (LPP) rats as determined by in vivo pineal microdialysis (14). Lights were off at 1 am and on at 11 am (within the shaded area). The diurnal pattern is reproducible in 100% of the animals examined, with slight variations in the level of melatonin among individual rats. There is no statistical difference between LPP and LPW rat strains in the level of pineal melatonin secretion.

synthesis is regulated at a number of different levels; for example, flashes of light in the night rapidly inactivate hormone synthesis by activation of proteosomal degradation of NAT (15); perhaps PINA participates in the nighttime regulation of NAT protein stability and therefore is only active in the night pineal. Alternatively, because little is known about pineal functions beyond its well-established duty as the body's source of melatonin, perhaps PINA plays a role in novel functions of the pineal that occur simultaneously with melatonin synthesis. In analogy to the role of ATP7B in copper loading of ceruloplasmin, PINA may help load nocturnally synthesized copper proteins in the pineal. PINA could also play a role in the concentration of copper within cellular compartments that may be necessary for the biosynthesis or processing of novel pineal secretion products. Clearly, an understanding of the role of PINA in the pineal may suggest novel, analogous functions for ATP7B in the liver.

7. PINA AND WILSON'S DISEASE

Wilson's disease (WD) is an autosomal recessive disorder resulting from mutations in the ATP7B gene. Patients with WD suffer from two main types of symptom: brain disorder and liver disease. For unknown reasons, the clinical presentation of WD patients span a broad spectrum; some patients suffer purely from liver disease, some from only brain disease, whereas others experience both types of symptom. Although patients do not pose eye complaints, the cornea of the eye is also affected in many patients, resulting in the hallmark brown discoloration of the cornea, which is very specific for neurological Wilson's disease, the Kayser-Fleischer ring.

The pathogenesis of WD is thought to result from a systemic overload of copper, which accumulates primarily in the three major targets of WD: the brain, eye, and liver. The etiological significance of copper is supported by the efficacy of treatments, which are principally aimed at chelation of free copper. Clearly, although multiple lines of experimental evidence have demonstrated that copper is toxic to hepatocytes and causes oxidative damage, it is less clearly established that copper is directly harmful to the neurons of the brain under normal circumstances. Could the case for copper toxicity in neurological WD be an oversimplification? Several pieces of evidence suggest that the brain disorder seen in WD is caused by more than simple copper overload. It is known, for example, that in human subjects with Wilson's disease and liver diseases, brain copper is elevated but there is sometimes no evidence of the characteristic neurological disorder. In cases of copper toxicity (Indian cirrhosis), there is clear liver failure, yet there have been no neurological symptoms described (16-18). LEC rats, the animal model of Wilson's disease with mutant ATP7B, have clearly elevated brain copper levels, yet fail to demonstrate neurological dysfunction (19,20). These observations temper the traditional notions that copper alone causes neurotoxicity in WD. Alternatively, other factors affecting the brain or required for proper brain function may be released in the absence of functional ATP7B and PINA.

Coincidentally, in addition to the pineal, the retina and pigment epithelium expresses PINA but not ATP7B. Whether PINA dysfunction in the eye causes the Kayser-Fleischer ring remains to be determined, but consideration must be given in the future for a specific role of PINA in the development of brain dysfunction in Wilson's disease, particularly in light of conflicting data on the effects of copper on the brain.

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