A new mechanism of neurodegeneration: a proinflammatory cytokine inhibits receptor signaling by a survival peptide - PubMed (original) (raw)

A new mechanism of neurodegeneration: a proinflammatory cytokine inhibits receptor signaling by a survival peptide

H D Venters et al. Proc Natl Acad Sci U S A. 1999.

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Abstract

Heightened expression of both a proinflammatory cytokine, tumor necrosis factor alpha (TNF-alpha), and a survival peptide, insulin-like growth factor I (IGF-I), occurs in diverse diseases of the central nervous system, including Alzheimer's disease, multiple sclerosis, the AIDS-dementia complex, and cerebral ischemia. Conventional roles for these two proteins are neuroprotection by IGF-I and neurotoxicity by TNF-alpha. Although the mechanisms of action for IGF-I and TNF-alpha in the central nervous system originally were established as disparate and unrelated, we hypothesized that the signaling pathways of these two cytokines may interact during neurodegeneration. Here we show that concentrations of TNF-alpha as low as 10 pg/ml markedly reduce the capacity of IGF-I to promote survival of primary murine cerebellar granule neurons. TNF-alpha suppresses IGF-I-induced tyrosine phosphorylation of insulin receptor substrate 2 (IRS-2) and inhibits IRS-2-precipitable phosphatidylinositol 3'-kinase activity. These experiments indicate that TNF-alpha promotes IGF-I receptor resistance in neurons and inhibits the ability of the IGF-I receptor to tyrosine-phosphorylate the IRS-2 docking molecule and to subsequently activate the critical downstream enzyme phosphatidylinositol 3'-kinase. This intracellular crosstalk between discrete cytokine receptors reveals a novel pathway that leads to neuronal degeneration whereby a proinflammatory cytokine inhibits receptor signaling by a survival peptide.

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Figures

Figure 1

Figure 1

IGF-I promotes survival (solid line) and antiphosphotyrosine-precipitable PI3-kinase activity (dotted line) of primary murine cerebellar neurons. Five-day-old cerebellar granule neurons were washed, cultured without serum for 24 h, and treated with increasing concentrations of IGF-I for 1 day. Neuronal survival was determined with the mitochondrial dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (23). At 10 ng/ml, IGF-I tended (mean ± SEM; P < 0.10) to increase survival of primary neurons. Neuronal survival was significantly increased with doses of 50–200 ng/ml of IGF-I (∗, P < 0.005; n = 3). Treatment with IGF-I for 5 min increased the activity of PI3-kinase at concentrations greater than 10 ng/ml (∗, P < 0.05; n = 3).

Figure 2

Figure 2

IGF-I inhibits apoptosis of cerebellar neurons, as determined by intracellular flow cytometry with terminal deoxynucleotide transferase-mediated d-UTP nick-end labeling (TUNEL). (A) Apoptosis of primary cerebellar neurons was measured in granule neurons cultured in medium, IGF-I (100 ng/ml), or 10% FBS with 25 mM KCl. A representative fluorescence histogram shows that treatment with IGF-I reduced the apoptotic cell population from 54% to 15%. Only 12% of neurons cultured with FBS underwent apoptosis. (B) Summary of three independent experiments with flow cytometry using TUNEL. Treatment with IGF-I reduced the apoptotic cell population from 51% ± 8% to 14% ± 4% (P < 0.01, n = 3), and similar results were observed with FBS.

Figure 3

Figure 3

IRS-2 is the primary docking molecule expressed in murine cerebellar granule neurons. One hundred micrograms of whole-cell lysates from neurons was electrophoresed and blotted with specific antibodies to either IRS-1 or IRS-2. As a positive control, FDCP myeloid progenitor cells were used that were maintained in recombinant murine IL-3 (0.25 units/ml) and 5% heat-inactivated horse serum (36). In this representative Western blot, we could barely detect IRS-1 protein in cerebellar granule neurons, although the 165-kDa IRS-1 protein was easily visible in lysates from FDCP cells. In contrast, the 185-kDa IRS-2 protein was easily detectable in the same whole-cell lysates from either cerebellar granule neurons or FDCP cells.

Figure 4

Figure 4

TNF-α reduces phosphotyrosine phosphorylation of IRS-2 in cerebellar granule neurons. (A) Representative Western blot of lysates from cerebellar granule neurons immunoprecipitated with an IRS-2 antibody and blotted with an antiphosphotyrosine antibody. Cerebellar granule neurons were cultured and treated with IGF-I, TNF-α, or both, as described above. IRS-2 was not constitutively tyrosine-phosphorylated in either granule neurons (medium) or control FDCP hematopoietic cells (data not shown) under basal conditions. Addition of IGF-I to cerebellar neurons or FDCP myeloid progenitor cells led to substantial phosphorylation on tyrosine of the IRS-2 protein. Pretreatment with TNF-α substantially inhibited the ability of IGF-I to tryosine-phosphorylate IRS-2 in cerebellar neurons, whereas TNF-α alone had no effect. (B) IRS-2 is uniformly expressed in granule neurons, regardless of treatment. Representative Western blot of lysates (100 μg) from cerebellar granule neurons cultured and treated with IGF-I, TNF-α, or both, as described above. (C) Densitometric summary of four independent Western blots shown in A. Tyrosine phosphorylation of the 185-kDa IRS-2 protein was increased 4.8 ± 0.6-fold with IGF-I treatment. Tyrosine phosphorylation was limited to a 1.6 ± 0.7-fold increase in the presence of both IGF-I and TNF-α (∗, P < 0.01).

Figure 5

Figure 5

TNF-α inhibits the ability of IGF-I to activate IRS-2-precipitable PI3-kinase activity and neuronal survival. (A) Representative autoradiogram of a thin layer chromatogram used to measure the amount of IRS-2-precipitable PI3-kinase enzymatic activity. Cerebellar granule neurons were treated as described in the text, and PI3-kinase activity was measured in cell lysates that were precipitated with an anti-IRS-2 antibody. This autoradiogram shows that activation of IRS-2-precipitable PI3-kinase by IGF-I is inhibited by pretreatment with TNF-α. (B) TNF-α inhibits the ability of IGF-I to activate IRS-2-associated PI3-kinase activity. Results of three independent experiments show that TNF-α consistently reduces IGF-I activation of PI3-kinase from 5.1 ± 1-fold to 2.7 ± 0.7-fold (P < 0.005; n = 3). TNF-α alone does not affect lipid phosphorylation. (C) TNF-α causes neuronal degeneration by blocking the ability of IGF-I to promote neuronal survival. Cerebellar granule neurons were treated with TNF-α in the presence or absence of IGF-I (100 ng/ml). Cell survival was measured 24 h later as described in Fig. 1 (n = 3). The survival-promoting ability of IGF-I was inhibited (∗, P < 0.05) by 50% with as little as 10 pg/ml TNF-α and almost fully blocked at a concentration of 100 pg/ml. At the highest concentrations of 1,000 and 10,000 pg/ml, TNF-α alone caused a moderate reduction (P < 0.01) in neuronal survival (6% ± 3% and 5% ± 2%, respectively) as compared with cerebellar granule neurons cultured in medium alone (13% ± 3%).

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