Pat Wals

Glial Fibrillary Acidic Protein Transcription Responses to Transforming Growth Factor‐β1 and Interleukin‐1β Are Mediated by a Nuclear Factor‐1‐Like Site in the Near‐Upstream Promoter

Abstract: Elevated expression of glial fibrillary acidic protein (GFAP) is associated with astrocyte activation during responses to injury in the CNS. Because transforming growth factor‐β1 (TGF‐β1) and interleukin‐1β (IL‐1β) are released during neural responses to injury and because these cytokines also modulate GFAP mRNA levels, it is of interest to define their role in GFAP transcription. The increases of GFAP mRNA in response to TGF‐β1 and decreases in response to IL‐1β were shown to be transcriptionally mediated in rat astrocytes transfected with a luciferase‐reporter construct containing 1.9 kb of 5′‐upstream rat genomic DNA. Constructs containing sequential deletions of the rat GFAP 5′‐upstream promoter identified a short region proximal to the transcription start (‐106 to ‐53 bp) that provides full responses to TGF‐β1 and IL‐1β. This region contains an unusual sequence motif with overlapping nuclear factor‐1 (NF‐1)‐ and nuclear factor‐κB (NF‐κB)‐like binding sites and homology to known TGF‐β response elements. Mutagenesis (3‐bp exchanges) in ‐70 to ‐68 bp blocked the induction of GFAP by TGF‐β1 and the repression by IL‐1β. Gel shift experiments showed that the DNA segment ‐85 to ‐63 bp was bound by a factor(s) in nuclear extracts from astrocytes. The concentrations of these DNA binding factors were increased by treatment of astrocytes with TGF‐β1 and decreased by IL‐1β. Binding of these nuclear factors was blocked by mutation of ‐70 to ‐68 bp. Despite homology to NF‐1 or NF‐κB binding sites in the GFAP promoter at segment ‐79 to ‐67 bp, anti‐NF‐κB or anti‐NF‐1 antibodies did not further retard the gel shift of the nuclear factors/DNA complex. Moreover, astrocytic nuclear proteins do not compete for the specific binding to NF‐1 consensus sequence. Thus, nuclear factors from astrocytes that bind to the ‐85‐ to ‐63‐bp promoter segment might be only distantly related to NF‐1 or NF‐κB. These findings are pertinent to the use of GFAP promoter constructs in transgenic animals, because cis‐acting elements in the GFAP promoter are sensitive to cytokines that may be elaborated in response to expression of transgene products.

Kainic Acid and Decorticating Lesions Stimulate the Synthesis of C1q Protein in Adult Rat Brain

Abstract: The first component of the classic complement cascade, C1q, was increased in whole rat brain after lesioning by intraperitoneally injected kainic acid (KA) (20‐fold, 3 days after KA) and in the striatum ipsilateral to unilateral decortication (fivefold, 10 days after decortication). C1q was measured after purification by chromatography and electrophoresis. De novo biosynthesis of C1q 3 days after KA was increased >10‐fold, as measured by the incorporation of [35S]methionine into C1q after incubation of brain slices from KA‐treated rats for 2 h. In parallel with these responses, KA induced fivefold increase of C1q bioactivity, as evaluated with C1q‐dependent hemolysis. The contribution of C1q from entrapped cerebrovascular blood was evaluated by the effects of perfusion and was minor relative to the increases of C1q in response to KA lesioning. These findings support the hypothesis that the C1q protein detected by immunocytochemistry in senile plaques of Alzheimer brains and in the hippocampus after deafferenting lesions is synthesized by resident brain cells.

Apolipoprotein J (clusterin) activates rodent microglia in vivo and in vitro

Apolipoprotein J (apoJ; also known as clusterin and sulfated glycoprotein (SGP)‐2) is associated with senile plaques in degenerating regions of Alzheimers disease brains, where activated microglia are also prominent. We show a functional link between apoJ and activated microglia by demonstrating that exogenous apoJ activates rodent microglia in vivo and in vitro. Intracerebroventricular infusion of purified human plasma apoJ (∼4 µg over 28 days) activated parenchymal microglia to a phenotype characterized by enlarged cell bodies and processes (phosphotyrosine immunostaining). In vitro, primary rat microglia were also activated by apoJ, with changes in morphology and induction of major histocompatibility complex class II (MHCII) antigen. ApoJ increased the secretion of reactive nitrogen intermediates in a dose‐dependent manner (EC50 112 nm), which was completely blocked by aminoguanidine (AG), a nitric oxide synthase inhibitor. However, AG did not block the increased secretion of tumor necrosis factor‐α by apoJ (EC50 55 nm). Microglial activation by apoJ was also blocked by an anti‐apoJ monoclonal antibody (G7), and by chemical cleavage of apoJ with 2‐nitro‐5‐thiocyanobenzoate. The mitogen‐activated protein kinase kinase and protein kinase C inhibitors PD98059 and H7 inhibited apoJ‐mediated induction of reactive nitrogen intermediate secretion from cultured microglia. As a functional measure, apoJ‐activated microglia secreted neurotoxic agents in a microglia–neuron co‐culture model. We hypothesize that ApoJ contributes to chronic inflammation and neurotoxicity through direct effects on microglia.