Temporal dynamics of glyoxalase 1 in secondary neuronal injury

Abstract

Background: Enhanced glycolysis leads to elevated levels of the toxic metabolite methylglyoxal which contributes to loss of protein-function, metabolic imbalance and cell death. Neurons were shown being highly susceptible to methylglyoxal toxicity. Glyoxalase 1 as an ubiquitous enzyme reflects the main detoxifying enzyme of methylglyoxal and underlies changes during aging and neurodegeneration. However, little is known about dynamics of Glyoxalase 1 following neuronal lesions so far.

Methods: To determine a possible involvement of Glyoxalase 1 in acute brain injury, we analysed the temporal dynamics of Glyoxalase 1 distribution and expression by immunohistochemistry and Western Blot analysis. Organotypic hippocampal slice cultures were excitotoxically (N-methyl-D-aspartate, 50 µM for 4 hours) lesioned in vitro (5 minutes to 72 hours). Additionally, permanent middle cerebral artery occlusion was performed (75 minutes to 60 days).

Results: We found (i) a predominant localisation of Glyoxalase 1 in endothelial cells in non-lesioned brains (ii) a time-dependent up-regulation and re-distribution of Glyoxalase 1 in neurons and astrocytes and (iii) a strong increase in Glyoxalase 1 dimers after neuronal injury (24 hours to 72 hours) when compared to monomers of the protein.

Conclusions: The high dynamics of Glyoxalase 1 expression and distribution following neuronal injury may indicate a novel role of Glyoxalase 1.

Figure 1. Time-dependent protein changes of Glo1 in control- and excitotoxically– lesioned OHSC from rats.

A Representative Western blot of Glo1 and the related β-actin signals for standardisation. Glo1 antibody marked two bands: one with a molecular mass of 23 kDa referring to the monomer and a second with a molecular mass of 46 kDa representing the dimer of Glo1. B Temporal dynamics of Glo1 (monomer, dimer) in control OHSC normalised to their related β-actin values. No significant changes for Glo1 monomer and dimer were found over the examined time frame. However, at all investigated time points significant differences between monomer and dimer levels were present (p<0.0001). C Alterations of Glo1 (monomer, dimer, CTR) after excitotoxically-lesion in a time-dependent manner as normalised against β-actin values. Subsequently, monomer and dimer levels of the lesioned-group were normalised and presented in relation to their respective time controls which were set arbitrarily to 100%. The Glo1 monomer showed a significant difference at 48 h compared to time control (*, p<0.05). Statistically differences for the Glo1 dimer were found at 24 h and 48 h post-injury (*, 24 h, p<0.0001; 48 h, p<0.001) compared to monomer and time control. Furthermore, a significant increase in dimer levels was determined from 12 h to 24 h after NMDA-application (+, p<0.05). D The alteration of the Glo1 dimer/monomer (ratio) in OHSC as a time-dependent phenomenon of excitotoxicty. We observed a significant increase in the ratio of NMDA-group from 1 h to 24 h after NMDA-treatment (+, p<0.05). Moreover, significant differences between control- and NMDA-group were found at 24 h and 48 h after NMDA exposition (*, p<0.0001).

Figure 2. Morphological Glo1 variations after NMDA-treatment.

A OHSC immunostained with Glo1 and NeuN. Appearance of small round Glo1 positive cells 24-application. These cells disappeared at 72 h and only astrocytic processes displayed Glo1 immunoreactivity. Scale bar: 50 µm. B Translocation of Glo1 from cytosol to cell membrane in neurons 12 h after NMDA treatment. OHSC stained with Glo1, the neuronal cell membrane marker N-Cadherin and DAPI for the nuclei. Scale bar: 20 µm. In the time-frame between 24 h and 48 h small round Glo1 positive cells appeared. Over time a progressive nuclear fragmentation and reduction in size (24 h, diameter = 7.86 µm±1.48; 48 h, diameter = 4.45 µm±0.94) was observed. Scale bar: 10 µm (24 h), 20 µm (48 h).

Figure 3. Glo1 is expressed in different cell types and underlies a time-dependent changes in immunoreactivity after pMCAO.

A Coronal sections of cerebral cortex of spontaneously hypertensive rats with pMCAO were stained for Glo1. Immunohistochemistry shows the injury- and time-dependent Glo1 immunoreactivity in the different stages of secondary neuronal injury. B Identification of Glo1 immunoreactive cells. Sections were subjected to immunofluorescence for Glo1, DAPI for the nuclei and typical markers for endothelial cells (75 min; laminin), neurons (3 d; NeuN) or astrocytes (60 d; GFAP). Scale bars: 300 µm (A), 10 µm (B; 75 min), 20 µm (B; 3 d) and 100 µm (B; 60 d).