Metallothionein (MT)-III: Generation of polyclonal antibodies, comparison with MT-I+II in the freeze lesioned rat brain and in a bioassay with astrocytes, and analysis of Alzheimer's disease brains

Metallothionein-III is a low molecular weight, heavy-metal binding protein expressed mainly in the central nervous system. First identified as a growt h inhibitory factor (GIF) of rat cortical neurons in vitro, it has subseque ntly been shown to be a member of the metallothionein (MT) gene family and renamed as MT-III. In this study we have raised polyclonal antibodies in ra bbits against recombinant rat MT-III (rMT-III). The sera obtained reacted s pecifically against recombinant zinc- and cadmium-saturated rMT-III, and di d not cross-react with native rat MT-I and MT-II purified from the liver of zinc injected rats. The specificity of the antibody was also demonstrated in immunocytochemical studies by the elimination of the immunostaining by p reincubation of the antibody with brain (but not liver) extracts, and by th e results obtained in MT-III null mice. The antibody was used to characteri ze the putative differences between the rat brain MT isoforms, namely MT-III and MT-III, in the freeze lesion model of brain damage, and for developi ng an ELISA for MT-III suitable for brain samples. In the normal rat brain, MT-III was mostly present primarily in astrocytes. However, lectin stainin g indicated that MT-III immunoreactivity was also present in microglia, mon ocytes and/or macrophages in the leptomneninges and lying adjacent to major vessels. In freeze lesioned rats, both MT-X+II and MT-III immunoreactiviti es increased in the ipsilateral cortex. The pattern of MT-III immunoreactiv ity significantly differed from that of MT-I+II, since the latter was evide nt in both the vicinity of the lesioned tissue and deeper cortical layers, whereas that of the former was located only in the deeper cortical layers. This suggests different roles for these MT isoforms, and indeed in a new bi oassay measuring astrocyte migration in vitro, rMT-III promoted migration t o a higher extent than MT-I+II. Thus, MT-III could not only affect neuronal sprouting as previously suggested, but also astrocyte function. Finally, M T-III protein levels of patients with Alzheimer's disease (AD) were, if any thing, increased when compared with similarly aged control brains, which wa s in agreement with the significantly increased MT-III mRNA levels of AD br ains.