Phosphotyrosine antibodies specifically label ameboid microglia in vitro and ramified microglia in vivo

Tillotson, Meryl Lee; Wood, John G.

doi:10.1002/glia.440020604

Cited by 39 publications

(25 citation statements)

References 24 publications

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“…No such aggregations of Mac-1-positive microglia were observed in nontransgenic littermates (Figure 1B). Similarly, using an antibody to phosphotyrosine, which is a specific marker for microglia in the central nervous system, 20,21 clusters of intensely stained microglia were observed in transgenic mice but not in littermate controls (Figure 1, C and D). In contrast to antiMac-1 staining, antibodies to phosphotyrosine revealed only weak staining of microglia in nontransgenic control mice ( Figure 1D).…”

Section: Activated Microglia Associated With Congophilic Plaques But mentioning

confidence: 84%

Association of Microglia with Amyloid Plaques in Brains of APP23 Transgenic Mice

Stalder¹,

Phinney²,

Probst³

et al. 1999

The American Journal of Pathology

304

201

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Microglia are a key component of the inflammatory response in the brain and are associated with senile plaques in Alzheimer's disease (AD). Although there is evidence that microglial activation is important for the pathogenesis of AD , the role of microglia in cerebral amyloidosis remains obscure. The present study was undertaken to investigate the relationship between ␤-amyloid deposition and microglia activation in APP23 transgenic mice which express human mutated amyloid-␤ precursor protein (␤PP) under the control of a neuron-specific promoter element. Light microscopic analysis revealed that the majority of the amyloid plaques in neocortex and hippocampus of 14-to 18-month-old APP23 mice are congophilic and associated with clusters of hypertrophic microglia with intensely stained Mac-1-and phosphotyrosinepositive processes. No association of such activated microglia was observed with diffuse plaques. In young APP23 mice , early amyloid deposits were already of dense core nature and were associated with a strong microglial response. Ultrastructurally , bundles of amyloid fibrils , sometimes surrounded by an incomplete membrane , were observed within the microglial cytoplasm. However , microglia with the typical characteristics of phagocytosis were associated more frequently with dystrophic neurites than with amyloid fibrils. Although the present observations cannot unequivocally determine whether microglia are causal , contributory , or consequential to cerebral amyloidosis , our results suggest that microglia are involved in cerebral amyloidosis either by participating in the processing of neuron-derived ␤PP into amyloid fibrils and/or by ingesting amyloid fibrils via an uncommon phagocytotic mechanism. In any case, our observations demonstrate that neuron-derived ␤PP is sufficient to induce not only amyloid plaque formation but also amyloid-associated microglial activation similar to that reported in AD. Moreover, our results are consistent with the idea that microglia activation may be important for the amyloid-associated neuron loss previously reported in these mice. (Am J Pathol 1999, 154:1673-1684)Substantial evidence supports the view that processing of the amyloid-␤ precursor protein (␤PP) and accumulation of the amyloid-␤ peptide (A␤) in the brain of Alzheimer's disease (AD) patients is crucial to the pathophysiology of the disease. 1,2 Senile amyloid plaques in AD brains are surrounded and infiltrated by activated microglia, which acquire an amoeboid morphology and express various proteins involved in the central nervous system inflammation. [3][4][5] The tight association of amyloid fibrils and microglia has suggested that microglia are somehow involved in either the formation or the phagocytosis of amyloid fibrils. 3,6 -10 Activation of microglia is thought to induce an inflammatory response in the central nervous system and to be a mediator of the amyloid-associated neurodegeneration in AD brain. 11,12 The involvement of inflammation in the progress of AD is underlined by clinical studies showing...

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Section: Activated Microglia Associated With Congophilic Plaques But mentioning

confidence: 84%

Association of Microglia with Amyloid Plaques in Brains of APP23 Transgenic Mice

Stalder¹,

Phinney²,

Probst³

et al. 1999

The American Journal of Pathology

304

201

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“…CD45 was localized using a well-characterized monoclonal antibody (Oncogene Science, Uniondale, N Y ) . Preparation and staining of rat microglial cultures was exactly as previously described (Tillotson and Wood, 1989b). Controls for CD45 were the use of several nonimmune sera in the primary solution.…”

Section: Materials and Methods Immunohistochemistrymentioning

confidence: 99%

Microglial tyrosine phosphorylation systems in normal and degenerating brain

Karp

Tillotson

Soria

et al. 1994

Glia

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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.

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“…brain (Tillotson and Wood, 1989;Karp et al, 1994). However, 15 min after ischemia, the immunoreactivity of phospho-PYK2, as well as of phosphotyrosine, was already increased.…”

Section: Immunolocalization Of Phosphorylated Pyk2 Within 1 Hr After mentioning

confidence: 99%

Cerebral Ischemia and Seizures Induce Tyrosine Phosphorylation of PYK2 in Neurons and Microglial Cells

Tian

Lev

2000

J. Neurosci.

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The nonreceptor tyrosine kinase PYK2 represents a stresssensitive mediator of c-Jun N-terminal kinase and p38 mitogenactivated protein kinase (MAPK) signaling pathways in many cell types. In the present study, we assessed the tyrosine phosphorylation of PYK2 under normal and pathological conditions in the CNS. We generated a polyclonal antibody that selectively recognizes tyrosine-phosphorylated PYK2 at its major autophosphorylation site. By using this antibody, we demonstrate that the phosphorylation profile of PYK2 after focal cerebral ischemia is biphasic. The first phase occurs within 1 hr, when most of the phospho-PYK2 immunoreactivity was observed in cortical neurons, whereas 24-72 hr after ischemia, a striking induction of phospho-PYK2 immunoreactivity was evident in microglia around the necrotic infarcted area. Double-immunostaining analysis using both anti-phospho-PYK2 antibody and antibody against the double-phosphorylated active form of p38MAPK revealed that the two phosphorylated protein kinases exhibit strikingly similar distribution patterns after ischemia. A short time after ischemia, phosphorylation of p38MAPK was evident in the cortical neurons as demonstrated by both immunohistochemistry and immunoblotting analysis, whereas 24-72 hr after ischemia, phospho-p38MAPK was found in activated microglia and colocalized with phospho-PYK2. In contrast to cortical neurons, basal phospho-PYK2 immunoreactivity was observed in hippocampal pyramidal neurons, which was markedly decreased after kainate acid-induced status epilepticus. However, 24 hr after the epileptic onset, a pronounced upregulation of PYK2 and phospho-PYK2 immunoreactivities was evident in microglial cells, as demonstrated by double-immunostaining with the microglial marker OX42. These results provide, for the first time, in situ localization of tyrosine-phosphorylated PYK2 in neuronal stress pathways in the adult rat brain and are consistent with the role of PYK2 as an upstream regulator of p38MAPK signaling cascades in response to stress signals.

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Phosphotyrosine antibodies specifically label ameboid microglia in vitro and ramified microglia in vivo

Cited by 39 publications

References 24 publications

Association of Microglia with Amyloid Plaques in Brains of APP23 Transgenic Mice

Association of Microglia with Amyloid Plaques in Brains of APP23 Transgenic Mice

Microglial tyrosine phosphorylation systems in normal and degenerating brain

Cerebral Ischemia and Seizures Induce Tyrosine Phosphorylation of PYK2 in Neurons and Microglial Cells

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