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Apart from the tremor phenotype, the most significant difference between Purα KO and wild type mice is the brain abnormalities: Brains of Purα KO mice were visibly enlarged and the weight was increased by 30% as compared to wild types. This is mainly due to the enlarged brain stem, whose weight increased by 200% according to the detailed brain analysis of Purα KO mice. Hippocampus and sensoric cortex of Purα KO mice were also enlarged but their weights decreased as compared to wild types. The enlargement of the cerebral cortex is accompanied by an increased surface.

A comparable phenotype has not been observed in any dysmyelination mutant mice, including shiverer or MLD mice. Moreover, the MLD mice displayed a normal but slightly reduced brain weight compared to wild type (Matthieu et al., 1984). However, such phenotype of enlarged brain is also observed in some other mice models: for example, megencephaly mice, IGF-1 (Insulin-like growth factor I) transgenic mice and the bcl-2 transgenic mice.

The reason for such an enlarged brain can be different, for example, the megencephaly mouse is characterized by an overall hypertrophy of the brain, and not by hyperplasia of particular cell types or by hypertrophy of a singular tissue compartment. Megencephaly also occurs in several acquired and inherited human diseases including Sotos syndrome, Ribinow syndrome, Canavan’s diseases, and Alexander disease. This defect can be distinguished from macrocephaly, an enlarged head, which usually occurs as a consequence of congenital

hydrocephalus. Compositional analysis of the brains of the megencephaly mice showed the water as a percentage of brain weight was not different between the two genotypes, excluding edema. Moreover, the ventricles of megencephaly mice were not dilated, ruling out hydrocephalus. Thus, larger brains in the megencephaly mutants are a consequence of increased cell size only. However, the enlarged brains of Purα knock-out mice are resulted from the increased cell mass as indicated by our new analysis (data not shown). Actually, this enlargement is only a local event, different from the situation in megencephaly mouse.

The IGF-1 transgenic mice have brains that are 55% larger than controls by approximately 2 months of age, with most brain structures affected. However, the increased brain size in transgenic mice results from both increased cell size and increased cell number (Carson et al., 1993). The overexpression of IGF-1 in Purα KO mice is not possible, since the largement in the brains of Purα KO mice is not correlated with increased cell number.

The brain size of bcl-2 transgenic mice increased by 50% as compared to wild type animals (Martinou et al., 1994). The bcl-2 gene has a powerful inhibitory action on naturally occurring cell death (NOCD) or apoptosis, which is a prominent feature of the developing nervous system. During this process, the expression of bcl-2 in neurons may determine whether a neuron dies or survives. bcl-2 overexpression reduces neuronal loss during the NOCD period, which leads to increased cell number of the nervous system. For instance, the facial nucleus and the ganglion cell layer of the retina had, respectively, 40% and 50% more neurons than normal. The structures most obviously enlarged are the brain stem and optic nerves (Martinou et al., 1994). These stems in the enlarged brains resemble those in Purα KO mice where the increased cell number is nonetheless not observed.

Our study shows that a deletion in a single gene, Purα, can have a dramatic effect on brain size and weight. It was shown that such an enlarged brain is due to the increased cell size but not increased cell number. However, Edema or hydrocephalus cannot be excluded.

The data of fresh wet brain weights showed: (i) The brains of the 60-66 day old Purα KO mice were significantly heavier than those of wild type; (ii) the brain weight of the heterozygotes resided exactly between the brain weights of the KO and wild type mice. However, the brain weights of 16-day old mice did not differ among the three genotypes. These results indicate that Purα KO mice demonstrate a significant increase in brain weight beginning from 16 days of age and continuing throughout at least to 66 days of age, while the normal adult brain weight in mice is achieved at approximately 2 weeks of age (Belknap et al., 1992). The further measurement of the brain weight after 66 day postnatally is not carried out and a further continuously increase in brain weight of the KO mice cannot be excluded.

We can also conclude that the tremor phenotype of the Purα KO mice is not due to the enlarged brain since the onset of tremor begins at postnatal day 12 (p12) while the enlarged brain forms definitively after postnatal day 16 (p16). Moreover, the heterozygotes also have enlarged brains but do not display tremor phenotype. These data suggest that the tremor phenotype might be independent of the enlarged brain and must be due to some other reasons, which are still unclear.

Histological staining of three areas (anterior commissure, facial nerve and pyramidal tract) of the brain of Purα KO mouse revealed increased lipid staining (Sudan black staining) as compared to wild type. This observation suggests an increased lipid level in the brains of Purα KO animals. If specific lipids are enriched in brain is unclear. The increased lipid level might be the reason of the brain hypertrophy of the Purα KO mice. Moreover, histological staining also showed slightly reduced labeling of the myelin sheaths in the brain of KO mouse. Whether it is caused by the reduced level of MBP in the beginning of myelination remains unclear. Altogether, the reduced myelin sheath and increased lipid level in brain suggest an possible changed compositions of the brains and this increased lipid level might affect signal transduction in nervous system, which possibly can lead to the tremor phenotype of the KO mice.