RANTES upregulation in the Alzheimer's disease brain: a possible neuroprotective role

Abstract

Numerous studies demonstrate inflammatory proteins in the brain and microcirculation in Alzheimer's disease (AD) and implicate inflammation in disease pathogenesis. However, emerging literature suggests that neuroinflammation can also be neuroprotective. The chemokine RANTES has been implicated in neurodegenerative diseases including AD. The objectives of this study are to determine the expression of RANTES in AD microvessels, its regulation in endothelial cells and its effects on neuronal survival. Our data show elevated expression of RANTES in the cerebral microcirculation of AD patients. Treatment of neurons in vitro with RANTES results in an increase in cell survival and a neuroprotective effect against the toxicity of thrombin and sodium nitroprusside. Oxidative stress upregulates RANTES expression in rat brain endothelial cells. Developing strategies to augment neuroprotection and diminish inflammatory activation of multifunctional mediators such as RANTES holds promise for the development of novel neuroprotective therapeutics in AD.

Fig. 1. AD-derived microvessels express and release high levels of RANTES

(a) Total RNA from brain microvessel from AD-derived (AD) and age matched controls (HC) were reverse transcribed and amplified using specific primers for RANTES. Data are normalized relative to GAPDH expression. Bar graph represents data from 7 AD samples and 7 control samples; ***p<0.001 vs. control. (b) Control (HC) and AD-derived microvessels (50 μg) were incubated in serum-free media supplemented with 1% LAH for 4 h. Microvessels were centrifuged and the supernatant analyzed by ELISA. Data are mean ± SD from 5 AD and 5 control patient samples performed in quadruplicate. The data are normalized to samples containing 1% LAH; *p<0.05 vs. control.

Fig. 1. AD-derived microvessels express and release high levels of RANTES

(a) Total RNA from brain microvessel from AD-derived (AD) and age matched controls (HC) were reverse transcribed and amplified using specific primers for RANTES. Data are normalized relative to GAPDH expression. Bar graph represents data from 7 AD samples and 7 control samples; ***p<0.001 vs. control. (b) Control (HC) and AD-derived microvessels (50 μg) were incubated in serum-free media supplemented with 1% LAH for 4 h. Microvessels were centrifuged and the supernatant analyzed by ELISA. Data are mean ± SD from 5 AD and 5 control patient samples performed in quadruplicate. The data are normalized to samples containing 1% LAH; *p<0.05 vs. control.

Fig. 2. Oxidative stress stimulates RANTES expression and release

(a) Rat brain endothelial cells were incubated with menadione (0.5 – 50 μM). Total RNA extracted was reverse-transcribed and amplified with RANTES primers. Data are representative of 3 separate experiments. (b) Rat brain endothelial cells were incubated with menadione (0.5 – 25 μM) and RANTES released into the supernatant quantified by ELISA; ***p<0.001 vs control. (c) Rat brain endothelial cells were incubated with H₂O₂ (1 – 500 μM). Total RNA extracted was reverse-transcribed and amplified with RANTES primers. Data are representative of 3 separate experiments. (d) Rat brain endothelial cells were incubated with H₂O₂ (1 – 500 μM) and RANTES released into the supernatant quantified by ELISA; ***p<0.001 vs. control. Each experiment was performed in triplicate.

Fig. 2. Oxidative stress stimulates RANTES expression and release

(a) Rat brain endothelial cells were incubated with menadione (0.5 – 50 μM). Total RNA extracted was reverse-transcribed and amplified with RANTES primers. Data are representative of 3 separate experiments. (b) Rat brain endothelial cells were incubated with menadione (0.5 – 25 μM) and RANTES released into the supernatant quantified by ELISA; ***p<0.001 vs control. (c) Rat brain endothelial cells were incubated with H₂O₂ (1 – 500 μM). Total RNA extracted was reverse-transcribed and amplified with RANTES primers. Data are representative of 3 separate experiments. (d) Rat brain endothelial cells were incubated with H₂O₂ (1 – 500 μM) and RANTES released into the supernatant quantified by ELISA; ***p<0.001 vs. control. Each experiment was performed in triplicate.

Fig. 2. Oxidative stress stimulates RANTES expression and release

(a) Rat brain endothelial cells were incubated with menadione (0.5 – 50 μM). Total RNA extracted was reverse-transcribed and amplified with RANTES primers. Data are representative of 3 separate experiments. (b) Rat brain endothelial cells were incubated with menadione (0.5 – 25 μM) and RANTES released into the supernatant quantified by ELISA; ***p<0.001 vs control. (c) Rat brain endothelial cells were incubated with H₂O₂ (1 – 500 μM). Total RNA extracted was reverse-transcribed and amplified with RANTES primers. Data are representative of 3 separate experiments. (d) Rat brain endothelial cells were incubated with H₂O₂ (1 – 500 μM) and RANTES released into the supernatant quantified by ELISA; ***p<0.001 vs. control. Each experiment was performed in triplicate.

Fig. 2. Oxidative stress stimulates RANTES expression and release

(a) Rat brain endothelial cells were incubated with menadione (0.5 – 50 μM). Total RNA extracted was reverse-transcribed and amplified with RANTES primers. Data are representative of 3 separate experiments. (b) Rat brain endothelial cells were incubated with menadione (0.5 – 25 μM) and RANTES released into the supernatant quantified by ELISA; ***p<0.001 vs control. (c) Rat brain endothelial cells were incubated with H₂O₂ (1 – 500 μM). Total RNA extracted was reverse-transcribed and amplified with RANTES primers. Data are representative of 3 separate experiments. (d) Rat brain endothelial cells were incubated with H₂O₂ (1 – 500 μM) and RANTES released into the supernatant quantified by ELISA; ***p<0.001 vs. control. Each experiment was performed in triplicate.

Fig. 3. LPS increases expression and release of RANTES in rat cultured brain endothelial cells

Cultured rat brain endothelial cells were grown to 85% confluent and treated with LPS (1 –500 ng) for 4 h. (a) Total RNA extracted was reverse-transcribed and amplified with RANTES primers. Data are representative of 3 separate experiments. (b) Release of RANTES into culture supernatant was quantified by ELISA; **p<0.01 vs control; ***p<0.001 vs control. Each experiment was performed in triplicate.

Fig. 3. LPS increases expression and release of RANTES in rat cultured brain endothelial cells

Cultured rat brain endothelial cells were grown to 85% confluent and treated with LPS (1 –500 ng) for 4 h. (a) Total RNA extracted was reverse-transcribed and amplified with RANTES primers. Data are representative of 3 separate experiments. (b) Release of RANTES into culture supernatant was quantified by ELISA; **p<0.01 vs control; ***p<0.001 vs control. Each experiment was performed in triplicate.

Fig. 4. Oxidatively modified LDLs stimulate the expression of RANTES in rat endothelial cells

LDL was modified with copper sulfate or HNE. Rat brain endothelial cells were treated with 10 μg/ml LDL, Ox-LDL or HNE-LDL for 24 h. RANTES released into the supernatant was determined by ELISA. Data are mean ± SD values for 3 separate experiments; **p<0.01 vs. LDL. Each experiment was performed in triplicate.

Fig. 5. Neuroprotective effect of RANTES on cerebral cortical cultures

Rat cortical cultures were incubated with or without RANTES (25 – 500 ng/ml) for 24 h. Cell viability was determined with the MTT assay. The number of viable cells without any RANTES treatment (control) were defined as 100 %. (a) Pretreatment with RANTES resulted in significant (p<0.001) cell survival compared to cells incubated without RANTES. (b) Rat cortical cultures pretreated with RANTES (25 – 500 ng/ml) for 24 h were incubated with 100 nM of thrombin for 18 h and cell viability determined; **p<0.01 vs control; ***p<0.001 vs control. (c) Rat cortical cultures pretreated with RANTES (25 – 500 ng/ml) for 24 h were incubated with 1 mM SNP for 4 h and cell viability determined; *p<0.05 vs control; ***p<0.001 vs control. Each experiment was performed in triplicate.

Fig. 5. Neuroprotective effect of RANTES on cerebral cortical cultures

Rat cortical cultures were incubated with or without RANTES (25 – 500 ng/ml) for 24 h. Cell viability was determined with the MTT assay. The number of viable cells without any RANTES treatment (control) were defined as 100 %. (a) Pretreatment with RANTES resulted in significant (p<0.001) cell survival compared to cells incubated without RANTES. (b) Rat cortical cultures pretreated with RANTES (25 – 500 ng/ml) for 24 h were incubated with 100 nM of thrombin for 18 h and cell viability determined; **p<0.01 vs control; ***p<0.001 vs control. (c) Rat cortical cultures pretreated with RANTES (25 – 500 ng/ml) for 24 h were incubated with 1 mM SNP for 4 h and cell viability determined; *p<0.05 vs control; ***p<0.001 vs control. Each experiment was performed in triplicate.

Fig. 5. Neuroprotective effect of RANTES on cerebral cortical cultures

Rat cortical cultures were incubated with or without RANTES (25 – 500 ng/ml) for 24 h. Cell viability was determined with the MTT assay. The number of viable cells without any RANTES treatment (control) were defined as 100 %. (a) Pretreatment with RANTES resulted in significant (p<0.001) cell survival compared to cells incubated without RANTES. (b) Rat cortical cultures pretreated with RANTES (25 – 500 ng/ml) for 24 h were incubated with 100 nM of thrombin for 18 h and cell viability determined; **p<0.01 vs control; ***p<0.001 vs control. (c) Rat cortical cultures pretreated with RANTES (25 – 500 ng/ml) for 24 h were incubated with 1 mM SNP for 4 h and cell viability determined; *p<0.05 vs control; ***p<0.001 vs control. Each experiment was performed in triplicate.