Carrasco Javier. Giralt Mercedes. Molinero Amalia. Penkowa Milena. Moos
Torben. Hidalgo Juan [a].
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, Journal of Neurotrauma 16(11) :1115-1129, 1999.
Abstract
Metallothionein-III is a low molecular weight, heavy-metal binding protein
expressed mainly in the central nervous system. First identified as a growth
inhibitory factor (GIF) of rat cortical neurons in vitro, it has subsequently
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 rabbits
against recombinant rat MT-III (rMT-III). The sera obtained reacted
specifically against recombinant zinc- and cadmium-saturated rMT-III, and did
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
preincubation of the antibody with brain (but not liver) extracts, and by the
results obtained in MT-III null mice. The antibody was used to characterize
the putative differences between the rat brain MT isoforms, namely MT-I+II
and MT-III, in the freeze lesion model of brain damage, and for developing an
ELISA for MT-III suitable for brain samples. In the normal rat brain, MT-III
was mostly present primarily in astrocytes. However, lectin
staining indicated that MT-III immunoreactivity was also present in
microglia, monocytes and/or macrophages in the leptomeninges and lying
adjacent to major vessels. In freeze lesioned rats, both MT-I+II and MT-III
immunoreactivities increased in the ipsilateral cortex. The pattern of MT-III
immunoreactivity significantly differed from that of MT-I+II, since the
latter was evident 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 bioassay measuring astrocyte
migration in vitro, rMT-III promoted
migration to a higher extent than MT-I+II. Thus, MT-III
could not only affect neuronal sprouting as previously suggested, but also
astrocyte function. Finally, MT-III protein levels of
patients with Alzheimer's disease (AD) were, if anything, increased when
compared with similarly aged control brains, which was in agreement with the
significantly increased MT-III mRNA levels of AD brains.
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