Differential expression of protein tyrosine kinase genes during microglial activation

Abstract

Protein tyrosine kinase (PTK) activity is abundant in microglia, but the PTKs that participate in their activation have not been identified. For these studies, we used three paradigms to characterize PTK expression during microglial activation: resting and activated microglia were bulk fractionated from the adult brain, cultured newborn microglia were treated with lipopolysaccharide (LPS) to model the transition from activated toward phagocytic microglia, and PTK expression was examined in activated microglia in situ after facial nerve axotomy. Two PCR-based strategies were used to show that 21 different PTK genes are expressed by rat brain microglia: 5 receptor PTKs, 10 nonreceptor PTKs, and 6 members of the src family. Seven of the 21 PTKs were examined in greater detail. Five PTK mRNAs (fgr, hck, fak, jak-2, and flk-1) increased expression across all three models of activation. We conclude that they represent key components in the cascades that participate in microglial activation. In contrast, expression of fes and fms correlated with stimuli that affect microglial proliferation. Four of the PTKs (hck, fgr, fes, and fms) are believed to be myeloid cell specific and were not expressed by cultured astrocytes. HCK and FAK protein were also not expressed in lysates of immature astrocytes and oligodendrocytes. Because of their putative specificity, these kinases represent potential targets for inhibitors of microglial activation. Because reactive microglia can exacerbate the severity of neurological diseases, the identification of specific kinases that participate in microglial activation represents an important advance toward the development of new therapeutics.

Characterization of an improved procedure for the removal of microglia from confluent monolayers of primary astrocytes

“…However, because microglia have been shown to exhibit heterogeneity for CD11b expression (Santambrogio et al, 2001), these results were confirmed by expression of c-fms mRNA. This transcript encodes the receptor for macrophage colony stimulating factor, which is also expressed by microglia but not astrocytes (Hao et al, 1990;Krady et al, 2002). The absence of both CD11b immunolabeling and c-fms expression after treatment with 75 mM LME provided independent confirmation of the effective removal of the surviving rogue microglia in Ara-Ctreated monolayers.…”

“…Microglia but not astrocytes have been reported to express the mRNA for the CSF-1 receptor, c-fms (Hao et al, 1990;Krady et al, 2002). Thus, expression of the c-fms transcript was assessed as a sensitive indicator for microglial contamination.…”

Lyn tyrosine kinase is required for P2X4 receptor upregulation and neuropathic pain after peripheral nerve injury

“…However, because the sequences surrounding the phosphorylation sites are highly similar among the SFKs (Thomas and Brugge, 1997), the possibility that the antibodies they used (Katsura et al, 2006) may cross-react with other SFKs, including Lyn, can not be excluded. SFKs other than the five CNS-type SFK members we examined, such as Hck and Fgr, have also been reported to be expressed in microglia in vitro (Krady et al, 2002), and thus, may be partly responsible for the microglial phospho-SFK-IR observed after nerve injury in lyn-deficient mice. In the future, by generating double-(or triple-) knockout mice for Lyn and other SFKs, and by studying their phenotypes, the roles of other SFKs in the residual active-SFK in dorsal horn microglia in lyn 2/2 mice will be determined.…”

The tyrosine kinase inhibitor AG126 restores receptor signaling and blocks release functions in activated microglia (brain macrophages) by preventing a chronic rise in the intracellular calcium level

“…IRAK is a serine/threonine kinase, rendering direct actions of the tyrphostin unlikely. We rather consider Fak, Fgr, Flk-1, Hck or Jak-2 as PTKs that change in microglia under the influence of LPS (Krady et al 2002). On the other hand, we already demonstrated that the interference of AG126 with microglial signaling and responses, e.g.…”