Phosphotyrosine and protein tyrosine phosphatase antibodies have been used to assess the distribution and potential functions of tyrosine phosphorylation systems in normal brain and cell cultures, as well as in a model of neural degeneration. Western blot and immunohistochemical analysis showed that a panel of antiphosphotyrosine antibodies recognizing different tyrosine phosphorylated substrates all selectively labeled ramified microglia in sections of brain tissue. This significantly extends our previous observation (GLIA 2:412-419, 1989) that a single, limited, phosphotyrosine antibody served as a histological marker for microglia. The present results show that tyrosine phosphorylation of a variety of substrates is quantitatively enriched in microglia compared to other neural cell types. We also show that the protein tyrosine phosphatase, CD45, is constitutively expressed by ramified microglia in vivo and by ameboid microglia in vitro. Thus, the major enzymes constituting tyrosine phosphorylation systems are present in normal microglia. Neuronal degeneration in the trigeminal nucleus, caused by introduction of the neurotoxic lectin, ricin, into the peripheral nerve is accompanied by a robust upregulation of phosphotyrosine signal in ramified microglial adjacent to the nucleus and in ameboid microglia in the degenerating nucleus. The presence of phosphotyrosine in ramified microglia is consistent with a role for tyrosine phosphorylation systems in the activation of microglia and in the signaling events accompanying conversion of resting microglia to the ameboid form.

Using an affinity-purified, polyclonal antibody to phosphotyrosine (Wang: Molecular and Cellular Biology 5:3640-3643, 1985) we have previously demonstrated that phosphotyrosine immunoreactivity is restricted to a population of multipolar GFAP-negative neuroglia in adult rat brain (Tillotson and Wood: Journal of Comparative Neurology 282:133-141, 1989) and retina (Tillotson and Wood: Journal of Cell Biology 107:724a, 1988). In this study, we show that the phosphotyrosine-immunoreactive cells are microglia. This conclusion is supported by numerous morphological and ultrastructural similarities between the phosphotyrosine-immunoreactive cells and microglia. Furthermore, phosphotyrosine co-localizes with the microglial-specific B4 isolectin of Bandeiraea simplifolia-1 lectin. Phosphotyrosine antibodies also stain ameboid microglia in primary cultures of neonatal rat brain. In addition, after 7 days in vitro, microglia are the only phosphotyrosine-immunoreactive element in the cultures. This temporal pattern of staining in vitro mimics the developmental progression of phosphotyrosine immunoreactivity in situ, in which a variety of structures stain during postnatal neural development (Tillotson and Wood: Journal of Comparative Neurology 282:133-141, 1989), but only microglia stain in mature brain. The significance of phosphotyrosine-containing proteins potentially expressed in a microglial-specific manner is discussed.

We have used antibodies against phosphotyrosine to probe 50-microns cerebellar sections from rats of various ages as well as sections of adult brainstem, cerebrum, and olfactory bulb to investigate the developmental appearance of this phosphorylation system as revealed by light microscopy. While the overall intensity of staining in the cerebellum was highest at 7 days, the pattern of staining in the adult is quite disparate from that seen in younger animals. From 10 to 21 days postnatal, staining is associated primarily with the white matter and/or the lower premigratory zone of the external granular layer and adjacent formative molecular layer. While the temporal and spatial appearance of phosphotyrosine immunoreactivity corresponds well to the established patterns of axonal growth in these areas, we cannot at the light level ascertain whether the immunoreactivity is intrinsic or extrinsic to the growing fibers. In animals 28 days and older, however, staining is restricted to a subpopulation of multipolar cells distributed throughout the cerebellum, as well as the olfactory bulb, cerebrum, and brainstem. The phosphotyrosine-positive cells in the adult cerebellum are not comparable to glial-fibrillary-acidic-protein-immunoreactive elements with regard to morphology or distribution, and they fail to colocalize with neuronal somata stained with anti-microtubule-protein-2 antibodies. While it appears that the radial fibers of the Bergmann glia in the external granular layer stain at 7 days, there is no staining detected in the mitotically active neuroblasts of this layer at any age. We conclude that in the immature cerebellum, the majority of tyrosine phosphorylation detectable by this method may be involved in the formation and growth of axonal processes.(ABSTRACT TRUNCATED AT 250 WORDS)

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