Reactive Astrocytosis in a Mouse Model of Chronic Polyamine Catabolism Activation

Abstract

Background: In the brain, polyamines are mainly synthesized in neurons, but preferentially accumulated in astrocytes, and are proposed to be involved in neurodegenerative/neuroinflammatory disorders and neuron injury. A transgenic mouse overexpressing spermine oxidase (SMOX, which specifically oxidizes spermine) in the neocortex neurons (Dach-SMOX mouse) was proved to be a model of increased susceptibility to excitotoxic injury.

Methods: To investigate possible alterations in synapse functioning in Dach-SMOX mouse, both cerebrocortical nerve terminals (synaptosomes) and astrocytic processes (gliosomes) were analysed by assessing polyamine levels, ezrin and vimentin content, glutamate AMPA receptor activation, calcium influx, and catalase activity.

Results: The main findings are as follows: (i) the presence of functional calcium-permeable AMPA receptors in synaptosomes from both control and Dach-SMOX mice, and in gliosomes from Dach-SMOX mice only; (ii) reduced content of spermine in gliosomes from Dach-SMOX mice; and (iii) down-regulation and up-regulation of catalase activity in synaptosomes and gliosomes, respectively, from Dach-SMOX mice.

Conclusions: Chronic activation of SMOX in neurons leads to major changes in the astrocyte processes including reduced spermine levels, increased calcium influx through calcium-permeable AMPA receptors, and stimulation of catalase activity. Astrocytosis and the astrocyte process alterations, depending on chronic activation of polyamine catabolism, result in synapse dysregulation and neuronal suffering.

Keywords: astrocyte processes; excitotoxicity; nerve terminals; oxidative stress; polyamines; spermine oxidase (SMOX).

Figure 1

Increased levels of ezrin and vimentin in Dach-SMOX cerebral cortex. Western blot analysis on cerebral cortex homogenate from Dach-SMOX and control mice using antibodies against ezrin and vimentin. (A) Aliquots (25 µg/lane) of total crude preparations, containing both synaptosomes and gliosomes, were submitted to 8% SDS-PAGE followed by Western blot for ezrin and vimentin. The membrane was stripped and re-probed for β-actin. Four controls, as well as four Dach-SMOX mice, were analyzed. Protein standard molecular weights are reported (kDa). (B,C) The relevant immunoreactive bands were quantified and normalized versus β-actin. Data are reported as filled squares (control) and filled triangles (Dach-SMOX). Mean and SD are also reported (n = 4). * p ≤ 0.05, according to Mann-Whitney test. (D) The fold increase for ezrin and vimentin in Dach-SMOX mice is shown. Data are reported as filled squares (vimentin) and filled triangles (ezrin). Mean and SD are also reported (n = 4).

Figure 2

Significant reduction of Spm content in Dach-SMOX cerebrocortical gliosomes. Put, Spd, and Spm content in gliosomes and synaptosomes from Dach-SMOX and control mice. For details, see Materials and Methods. Filled circles represent the PA content (pmol/µg protein) in control (black) and Dach-SMOX (white) gliosomes (A) and synaptosomes (B). Mean and SD are reported (n = 4). * p < 0.05 according to one-way ANOVA test and post hoc test Tuckey.

Figure 3

AMPA receptor activation-evoked [Ca²⁺]_i increase in Dach-SMOX cerebrocortical gliosomes and synaptosomes. CG-loaded particles (gliosomes or synaptosomes) from control (○) and Dach-SMOX (●) mice were treated with 100 µM CPW399 for the indicated time at 37 °C. CG-dependent fluorescence was monitored every 10 s from 0 to 600 s. [Ca²⁺]_i increase is expressed as “increase of fluorescence”, which is the difference between the CG-dependent fluorescence of the stimulated samples and the ones of the vehicle-treated samples, both measured at each recording time and subtracted by the one measured at the starting time. (A) Data are means ± SEM from three (●) or four (○) independent experiments in triplicate. (B) Data are means ± SEM from three (●,○) independent experiments in triplicate. * p ≤ 0.05, according to Mann–Whitney test.

Figure 4

Antagonism by NASPM of the [Ca²⁺]_i influx evoked by AMPA receptor activation in Dach-SMOX cerebrocortical gliosomes and synaptosomes. CG-loaded particles (gliosomes or synaptosomes) were treated with 100 µM CPW399 in the absence (●) or in the presence (○) of the selective antagonist of GluA2-lacking AMPA receptor NASPM (50 µM) for the indicated time at 37 °C. CG-dependent fluorescence was monitored every 10 s from 0 to 240 s. [Ca²⁺]_i increase is expressed as “increase of fluorescence”. The areas reported in each upper panel were quantified to estimate the calcium influxes. (A) [Ca²⁺]_i increase (upper panel) and calcium influx (lower panel) in gliosomes from control mice. Data are means ± SEM from four (●) independent experiments in triplicate. NA, not assessed. (B) [Ca²⁺]_i increase (upper panel) and calcium influx (lower panel) in gliosomes from Dach-SMOX mice. Data are means ± SEM from three (●,○) independent experiments in triplicate. (C) [Ca²⁺]_i increase (upper panel) and calcium influx (lower panel) in synaptosomes from control mice. Data are means ± SEM from six (●) or five (○) independent experiments in triplicate. (D) [Ca²⁺]_i increase (upper panel) and calcium influx (lower panel) in synaptosomes from Dach-SMOX mice. Data are means ± SEM from seven (●, ○) independent experiments in triplicate. * p ≤ 0.05, according to Mann–Whitney test.

Figure 5

Analysis of catalase activity in Dach-SMOX cerebrocortical gliosomes and synaptosomes. The specific activity of catalase (mmoles of decomposed H₂O₂ per min per mg protein) was quantified by spectrophotometric assay in brain cortex (A) gliosomes and (B) synaptosome from both control and Dach-SMOX mice. Data are reported as filled squares (control) and filled triangles (Dach-SMOX). Mean and SD of experiments performed in quadruplicate on five animals per group are also reported. ** p ≤ 0.01, according to Mann–Whitney test.