CSF from Parkinson disease patients differentially affects cultured microglia and astrocytes.

BACKGROUND: Excessive and abnormal accumulation of alpha-synuclein (α-synuclein) is a factor contributing to pathogenic cell death in Parkinson's disease. The purpose of this study, based on earlier observations of Parkinson's disease cerebrospinal fluid (PD-CSF) initiated cell death, was to determine the effects of CSF from PD patients on the functionally different microglia and astrocyte glial cell lines. Microglia cells from human glioblastoma and astrocytes from fetal brain tissue were cultured, grown to confluence, treated with fixed concentrations of PD-CSF, non-PD disease control CSF, or control no-CSF medium, then photographed and fluorescently probed for α-synuclein content by deconvolution fluorescence microscopy. Outcome measures included manually counted cell growth patterns from day 1-8; α-synuclein density and distribution by antibody tagged 3D model stacked deconvoluted fluorescent imaging. RESULTS: After PD-CSF treatment, microglia growth was reduced extensively, and a non-confluent pattern with morphological changes developed, that was not evident in disease control CSF and no-CSF treated cultures. Astrocyte growth rates were similarly reduced by exposure to PD-CSF, but morphological changes were not consistently noted. PD-CSF treated microglia showed a significant increase in α-synuclein content by day 4 compared to other treatments (p ≤ 0.02). In microglia only, α-synuclein aggregated and redistributed to peri-nuclear locations. CONCLUSIONS: Cultured microglia and astrocytes are differentially affected by PD-CSF exposure compared to non-PD-CSF controls. PD-CSF dramatically impacts microglia cell growth, morphology, and α-synuclein deposition compared to astrocytes, supporting the hypothesis of cell specific susceptibility to PD-CSF toxicity.

Studies of the expression of DNA repair related genes

This dissertation examines the biological functions and the regulation of expression of DNA ligase I by studying its expression under different conditions.^ The gene expression of DNA ligase I was induced two- to four-fold in S-phase lymphoblastoid cells but was decreased to 15% of control after administration of a DNA damaging agent, 4-nitroquinoline-1-oxide. When cells were induced into differentiation, the expression level of DNA ligase I was decreased to less than 15% of that of the control cells. When the gene of DNA ligase I was examined for tissue specific expression in adult rats, high levels of DNA ligase I mRNA were observed in testis (8-fold), intermediate levels in ovary and brain (4-fold), and low levels were found in intestine, spleen, and liver (1- to 2-fold).^ In confluent cells of normal skin fibroblasts, UV irradiation induced the gene expression of DNA ligase I at 24 and 48 h. The induction of DNA ligase I gene expression requires active p53 protein. Introducing a vector containing the wild type p53 protein in the cells caused an induction of the DNA ligase I protein 24 h after the treatment.^ Our results indicate that, in addition to the regulation by phosphorylation/dephosphorylation, cellular DNA ligase I activity can be regulated at the gene transcription level, and the p53 tumor suppresser is one of the transcription factors for the DNA ligase I gene. Also, our results suggest that DNA ligase I is involved in DNA repair as well as in DNA replication.^ Also, as an early attempt to clone the human homolog of the yeast CDC9 gene which has been shown to be involved in DNA replication, DNA repair, and DNA recombination, we have identified a human gene with mRNA of 1.7 kb. This dissertation studies the gene regulation and the possible biological functions of this new human gene by examining its expression at different stages of the cell cycle, during cell differentiation, and in cellular response to DNA damage.^ The new gene that we recently identified from human cells is highly expressed in brain and reproductive organs (BRE). This BRE gene encodes an mRNA of 1.7-1.9 kb, with an open reading frame of 1,149 bp, and gives rise to a deduced polypeptide of 383 amino acid residues. No extensive homology was found between BRE and sequences from the EMBL-Gene Banks. BRE showed tissue-specific expression in adult rats. The steady state mRNA levels were high in testis (5-6 fold), ovary and brain (3-4 fold) compared to the spleen level, but low in intestine and liver (1-2 fold). The expression of this gene is responsive to DNA damage and/or retinoic acid (RA) treatment. Treatment of fibroblast cells with UV irradiation and 4-nitroquinoline-1-oxide caused more than 90% and 50% decreases in BRE mRNA, respectively. Similar decreases in BRE expression were observed after treatment of the brain glioma cell line U-251 and the promyelocytic cell line HL-60 with retinoic acid. (Abstract shortened by UMI). ^

Implication of bFGF signaling in lung carcinogenesis

A Western Array Screening system in conjunction with an in vitro lung carcinogenesis model, which consists of human bronchial epithelial (HBE) cells representing normal (NHBE), immortalized (BEAS-2B and 1799), transformed (1198), and tumorigenic (1170-I) was used to test the hypothesis that lung carcinogenesis involves specific changes in signaling proteins. Forty six proteins whose expression was upregulated by >2 fold and 23 proteins whose expression was downregulated by >2 fold in 1170-I compared to NHBE cells were identified. The levels of six proteins including bFGF (both intracellular and secreted), Akt and p70s6K in the PI3KJp70s6K pathway and the bFGF receptor (FGFR1) were upregulated in different stages of lung carcinogenesis. Akt activity and phospho-p70s6K were also increased in 1170-I compared to NHBE cells, suggesting that PI3K/p70s6K pathway is activated during lung carcinogenesis. bFGF treatment stimulated the growth of the 1170-I cells. Both tyrosine phosphorylation of FGFR1 and cell growth were inhibited in 1170-I cells after overexpression of dominant-negative(DN) FGFR1. Growth inhibition involved a G2 arrest related to decreased cdc2 activity, cdc25C downregulation, Wee1, p21(WAF1) and p27(Kip1) upregulation. Apoptosis was observed in tumorigenic but not in normal cells after overexpression of DNFGFR1. Confluent NHBE cells, were much less sensitive to the growth inhibition by DNFGFR1 compared to other cell lines analyzed. bFGF increased phospho-Akt and phospho-p70s6K in 1170-I cells. The Akt inhibitor LY294002 and the p70s6K inhibitor rapamycin inhibited bFGF-stimulated cell growth in 1170-I cells. Both agents downregulated the bFGF-induced increase in S phase by inducing G1 arrest. Also, LY294002 inhibited bFGF increased phospho-Akt, while both LY294002 and rapamycin inhibited bFGF increased phospho-p70s6K. Thus, cell proliferation stimulated by bFGF in 1170-I cells was at least partially mediated by PI3K/p70s6K pathway. Hsp90 was upregulated by bFGF in 1170-I cells. Its inhibitor geldanamycin inhibited the bFGF-stimulated growth via inducing apoptosis and G2 arrest through decreases in cdc2 expression/activity and p21 upregulation, and decreased Akt/phospho-Akt, p70s6K/phospho-p70s6K and Bad. Hsp90, p70s6K and Bad were found in the same complex, which may be important for signaling cell survival. Taken together, our study suggests that bFGF signaling, especially PI3K/p70s6K pathway, is important for lung carcinogenesis. ^