CONTEXTO SOCIOPOLÍTICO

4.3.1 Wasted

The gene that causes the wasted phenotype is likely to be expressed at least in the CNS and the lymphoid tissues, since this phenotype is associated with neurological and immunological abnormalities. The reader is reminded that wasted mice are recognised at about 21 days of age by symptoms which are characteristic of neurologic abnormalities. These include tremor and uncoordinated body movements which lead to progressive paralysis and death before 30 days of age (Shultz et a!., 1982). In addition, the wasted phenotype is associated with a severe wasting syndrome marked by loss of body weight, lymphoid hypoplasia of the spleen, the thymus, the mesenteric lymph nodes (MLN) and the Peyer’s patches (Shultz et a!., 1982, Kaiserlian et a!., 1986). Furthermore, this gene is very likely to be expressed postnatally.

This study demonstrated that the only known gene that is probably coincident with wst is the a4 subunit gene of the neural nicotinic acetylcholine receptor, Acra4. It is, therefore, worth considering the credentials of this gene as a candidate gene for wasted.

The nicotinic acetylcholine receptor (nAchR) subunits are of two types, a and p. The receptor itself is thought to be a pentamer of the type azps, although some alpha subunits could form functional homo-pentamers in vivo (reviewed in Sargent, 1993). Eight alpha (a2-a9) and three beta (P2-p4) subunit cDNAs have been isolated in the rat (Elgoyhen eta!., 1994 Sargent, 1993). Nothing is known about the structure or expression pattern of the Acra4 gene in the mouse. Its rat homologue codes for at least four transcripts (Goldman et a/., 1987). Three of these have been cloned whereas a fourth one was postulated on the basis of SI Nuclease protection experiments (Goldman et a!., 1987; this report describes two of these transcripts; the sequence of a third cDNA was recently submitted by these authors; EMBL Accession Number L31620). It is thought that these transcripts result from differential splicing of the a4 gene primary transcript (Goldman et a/., 1987). The rat a4 subunit gene is expressed in various areas of the central nervous system, including the spinal cord, thalamus, hypothalamus and brain stem (Goldman et a/., 1987). It is likely that Acra4 has the same pattern of expression in the mouse and could also code for multiple isoforms.

Could this gene be responsible for the neurological defects apparent in wasted mice? The answer to this is based only on circumstantial evidence. Wasted mice share features with mice homozygous for the neurological mutation oscillator (of) the genetic basis of which was recently established (Buckwalter et al., 1994). Affected ot/ot homozygotes demonstrate fine motor tremor at about 2 weeks of age, the severity of which increases daily leading to death by day 23 (Buckwalter at a/., 1994). The cause of this phenotype is a seven nucleotides deletion in the a l subunit of the glycine receptor subunit which abolishes the function and possibly assembly of the receptor (Buckwalter at a/., 1994). Glycine is a neurotransmitter whose receptor, an Œ2p3 pentamer, is a member of the ligand-gated ion channel receptor super-family,

which includes the acetylcholine receptor and type A gamma-aminobutiric acid (GABAa) receptors (reviewed in Betz, 1992). As the glycine receptor appears to function in the same way as acetylcholine receptors, as shown by in vivo studies using Xanopus oocytes (Betz, 1992; Sargent, 1993), it could be argued that a defect in the acetylcholine receptor could produce a similar phenotype like that caused by defects in the glycine receptor. Would we expect the acetylcholine receptor subunit genes to have a temporal expression pattern which would coincide with the onset of the wasted phenotype (21 days)? To my knowledge, there are no studies which examine the temporal expression pattern of the nAchR subunits genes, and any suggestion is again speculative. The expression of the a l subunit of the glycine receptor in the rat increases after birth reaching high levels by postnatal day 15, whereas the expression of the a2 subunit decreases steadily after birth reaching undetectable levels by day 15 (Malosio at a/., 1991). A similar developmental switch operating for nAch receptors could not be ruled out.

Could a defect in Acra4 lead to lymphoid hypoplasia of the peripheral lymphocytes and regression of the lymphoid organs? To my knowledge, no expression studies have been carried out in mice to test for the presence of neuronal nAchR on the surface of lymphocytes. Initial studies, carried out in the rat, demonstrated that low affinity nicotinic Ach receptors are present on the cell membrane of rat lymphocytes and thymocytes (Maslinski at a/., 1980 and 1987; Morgan at a/., 1984). Recently, it was demonstrated that human thymocytes (and not peripheral lymphocytes) express at least three genes which code for the a3, a5 and

p4 neuronal nAchR subunit genes (Mihovilovic and Roses, 1993). The a4 gene was not tested in this study but the first indications are that it is also expressed in the thymus (M. Mihovilovic, personal communication). Interestingly, studies in the rat showed that both T and B lymphocytes bear nicotinic receptors (Maslinski at a/., 1987). Activation of either of these receptors initiates mitosis leading to maturation of thymocytes (Maslinski at a/., 1987; Bulloch and Radojcic, 1989). These and other receptors, such as p-adrenergic ones, are thought to establish the link between the

because of low levels of IgA in wstMst homozygotes (Kaiserlian et al., 1985). It is important that in the rat gut, the IgA baseline production can be diminished by the muscarinic antagonist atropine, whereas the cholinergic (i.e. binds acetylcholine receptors) agonist carbachol can augment IgA levels (Wilson at a/., 1982). These studies do at least indicate that the lymphoid tissues of the mouse could express the Acra4 gene. They also suggest that the neurotransmitter acetylcholine is involved in the maturation process of lymphocytes and it is conceivable that a defect in this process would lead to peripheral lymphoid hypoplasia caused by a primary defect in the sites of lymphocyte maturation.

The recent finding that mice homozygous for a null mutation in the p2 subunit gene which abolishes the nAchR function are viable, mate normally and show no obvious signs of neurological abnormalities (Picciotto at a/., 1995) would argue against an association between neurological abnormalities and defective nAchR. Furthermore, a nonsense mutation in the human a4 subunit gene is associated with Benign Familial Neonatal Convulsions (BFNC; Beck at a/., 1994). BNFC is an autosomal dominant epilepsy which manifests within the first 3 days of life, and 90% of affected adults show no symptoms of epilepsy. So, abolishing the function of at least one isoform of the nAchR in human is not associated with neurological or immunological abnormalities similar to those seen in wasted mice. In human, the p2 subunit gene is not expressed in lymphoid tissues (Mihovilovic and Roses, 1993) and this could perhaps explain the absence of lymphoid hypoplasia in p Z /p T mice.

Whether Acra4 proves to be allelic to wst remains to be seen. From the point of view of testing for mutations in this gene, the single recombination event which occurred within this gene offers an excellent practical advantage, having effectively excluded 90% of the coding sequence from the candidate region (always judging from the structure of the rat cDNAs). If this gene does prove to be allelic to wst, then wasted mouse will make an excellent model for studying the interaction of the CNS and the immune system in postnatal development.

Recently, the gene for the pre-existing (p) nuclear factor for activated T cells (NFATp) has been assigned to band H of mouse chromosome 2 and human chromosome 20q13.2^ q13.31 by FISH analysis. This gene is expressed in the brain, heart, thymus and spleen (Northrop at a!., 1994). NFTAp is a multisubunit DNA-binding transcription factor present in the cytosol of unstimulated T cells which enters the nucleus upon T cell activation. It binds to several genes involved in T cell activation such as the IL-2 gene (McCaffrey at al., 1993 and references therein). On the basis of its expression pattern and its physical position, NFTAp was suggested as a candidate gene for wasted (Li at al., 1995). NFTAp maps proximal to band H4 (Li at al., 1995) and, therefore, is highly unlikely to be the wst gene.

4.3.2 Ragged

Very few studies have addressed the pathology of R ah or Ra/Ra mice. Given the effects that Ra has on hair growth, the product of this locus would be expected to be expressed in epidermal tissue. Although Acra4 is coincident with Ra, it seems unlikely that Acra4 would be the basis of the ragged phenotype (Abbott et al., 1994). The product of the Ra locus might be expressed in the kidneys and may be involved in ion transport for Ra/Ra homozygotes develop oedema which could be caused by malfunction of the kidneys or reduced renal mass (Abbott at a/., 1994). The three mutant alleles now identified at this locus {Ra, Ra®** and Ra'^ ) should improve the chances of identifying the causative mutations, particularly in view of the differences in severity of the phenotype associated with the Ra°^ allele compared to the other two alleles (Abbott at a/., 1994).