Soluble iron accumulation induces microglial glutamate release in the spinal cord of sporadic amyotrophic lateral sclerosis

Abstract

Previous studies on sporadic amyotrophic lateral sclerosis (SALS) demonstrated iron accumulation in the spinal cord and increased glutamate concentration in the cerebrospinal fluid. To clarify the relationship between the two phenomena, we first performed quantitative and morphological analyses of substances related to iron and glutamate metabolism using spinal cords obtained at autopsy from 12 SALS patients and 12 age‐matched control subjects. Soluble iron content determined by the Ferrozine method as well as ferritin (Ft) and glutaminase C (GLS‐C) expression levels on Western blots were significantly higher in the SALS group than in the control group, while ferroportin (FPN) levels on Western blots were significantly reduced in the SALS group as compared to the control group. There was no significant difference in aconitase 1 (ACO1) and tumor necrosis factor‐alpha (TNFα)‐converting enzyme (TACE) levels on Western blots between the two groups. Immunohistochemically, Ft, ACO1, TACE, TNFα, and GLS‐C were proven to be selectively expressed in microglia. Immunoreactivities for FPN and hepcidin were localized in neuronal and glial cells. Based on these observations, it is predicted that soluble iron may stimulate microglial glutamate release. To address this issue, cell culture experiments were carried out on a microglial cell line (BV‐2). Treatment of BV‐2 cells with ferric ammonium citrate (FAC) brought about significant increases in intracellular soluble iron and Ft expression levels and conditioned medium glutamate and TNFα concentrations. Glutamate concentration was also significantly increased in conditioned media of TNFα‐treated BV‐2 cells. While the FAC‐driven increases in glutamate and TNFα release were completely canceled by pretreatment with ACO1 and TACE inhibitors, respectively, the TNFα‐driven increase in glutamate release was completely canceled by GLS‐C inhibitor pretreatment. Moreover, treatment of BV‐2 cells with hepcidin resulted in a significant reduction in FPN expression levels on Western blots of the intracellular total protein extracts. The present results provide in vivo and in vitro evidence that microglial glutamate release in SALS spinal cords is enhanced by intracellular soluble iron accumulation‐induced activation of ACO1 and TACE and by increased extracellular TNFα‐stimulated GLS‐C upregulation, and suggest a positive feedback mechanism to maintain increased intracellular soluble iron levels, involving TNFα, hepcidin, and FPN.

Astrocytes release glutamate via cystine/glutamate antiporter upregulated in response to increased oxidative stress related to sporadic amyotrophic lateral sclerosis

“…This investigation was carried out on spinal cords obtained at autopsy from 12 sporadic ALS patients (age at death: 40‐83 [mean 65.9] years; sex: three men and nine women) and age‐matched control subjects (age at death: 42‐87 [mean 65.5] years; sex: seven men and five women), with P > 0.05 in age at death between the two groups. These cases were the same as those in our recent report 7 . Their clinical manifestation records other than age at death, sex, and disease duration were unavailable.…”

“…Thereafter, riluzole, an inhibitor of glutamate release from the glutamatergic nerve terminal, was developed for ALS therapy, based on the hypothesis that the potential major source of glutamate release in the synaptic cleft between the upper and lower motor neurons may be the upper motor neurons 1 . We also provide in vivo and in vitro evidence for microglial glutamate release promoted by aconitase‐1 activation and glutaminase C upregulation in response to increased soluble iron content in sporadic ALS spinal cords, 7 suggesting that excessive synaptic glutamate in this disease is derived from non‐neuronal cells. This has something in common with the bitter fact that efficacy of riluzole is limited 1 …”

“…These cases were the same as those in our recent report. 7 Their clinical manifestation records other than age at death, sex, and disease duration were unavailable. Each autopsy was performed with informed consent.…”

“…1 Increased oxidative stress in sporadic ALS patients is evidenced by several observations that the neuroglial cells contain an excess of oxidative modification products such as nitrotyrosine 2 , 8-hydroxy-2 0 -deoxyguaosine (OH-dG), 3 4-oxo-2-nonenal-dG (ONE-dG), 4 4-hydroxy-2-nonenal-histidine (HNEH), crotonaldehyde-lysine (CRAL), N ε -(carboxymethyl)lysine (CML), pentosidine, 5 and 4-hydroxy-2-hexenal-histidine (HHEH) 6 ; however, the essential cause of increased oxidative stress in this disease remains unknown. We recently demonstrated a significant increase in soluble iron content in sporadic ALS spinal cords, 7 which may generate increased oxidative stress via Fenton reaction, 8 although the major entry route of a large amount of soluble iron into the CNS has not yet been identified.…”

Senescent Microglia: The Key to the Ageing Brain?

“…This coincided with motor neuron loss in the model organism. Soluble iron is once again implicated in neurodegeneration, as its intracellular accumulation in microglia has been suggested to enhance activation of aconitase 1 (ACO1) and tumour necrosis factor α converting enzyme (TACE), leading to increased TNF-α stimulation of glutaminase-C (GLS-C), with resultant induction of glutamate release by microglia, contributing to excitotoxicity [ 161 ]. TNF-α, IL-1β, MMP12, and several other genes associated with a senescent phenotype have been found to be increased in ALS brains, again indicative of the potential for the involvement of microglial senescence in pathology [ 47 ].…”