Increased glutathione levels in neurochemically identified fibre systems in the aged rat lumbar motor nuclei

Abstract

The spinal cord motor nuclei have been the focus of a number of investigations exploring neurodegenerative mechanisms, e.g. excitotoxicity mediated by glutamate and oxidative stress. Here, high-resolution quantitative post-embedding immunocytochemistry with antibodies to oxidized and reduced glutathione (GSH), an ubiquitously expressed scavenger of free radicals, was used to examine if GSH synthesis is upregulated pre- and/or postsynaptically in the lumbar motor nuclei of aged (30 month old) rats. The purpose was, moreover, to resolve the extent of correlation between GSH expression, transmitter identity and degenerative changes. Tissue from young adult rats was co-processed for comparison. The quantitative immunogold analysis revealed an increase in GSH-immunoreactivity in both pre- and postsynaptic compartments in the lumbar motor nuclei of aged rats. Presynaptically, the enrichment of GSH-immunoreactivity was seen in axonal boutons of normal appearance, and was furthermore restricted to the extra-mitochondrial compartment. Postsynaptically, the aged rats disclosed, in comparison with young adults, higher values for GSH-immunoreactivity both over mitochondria (+49%) and cytoplasmic matrix (+130%). When analysing the transmitter identity of the bouton profiles, it turned out that close to 50% of all glutamate-immunoreactive boutons in the aged rats contained very high levels (> 40 gold particles/microm2) of GSH-immunoreactivity. Strong GSH-immunoreactivity was also a typical feature of a subset of axon terminal- and axon fibre-like profiles in the aged rat that showed signs of axon dystrophy and degeneration. When comparing with normally appearing axon fibre profiles located in close vicinity, the population of aberrant axons had higher average levels of glutamate-immunoreactivity (+93%), and lower average levels of glycine-immunoreactivity (-88%). No difference was seen regarding the levels of GABA. The results of this study lend support to the idea that aging in the spinal cord motor nuclei is associated with an increased oxidative stress and indicate that different transmitter systems are differentially affected by the degenerative process.

Comparison of GAD65 and 67 Immunoreactivity in the Lumbar Spinal Cord Between Young Adult and Aged Dogs

“…We could not get any data for aged-related change in GAD65 or 76. However, there is a report that Glutathione (gamma-glutamylcysteinylglycine), a neuromodulator at glutamate receptors, levels were significantly increased in neurochemically identified fibre systems in the aged rat lumbar motor nuclei [40], suggesting that aging in the spinal cord motor nuclei is associated with an increased oxidative stress.…”

Visualization of the compartmentalization of glutathione and protein‐glutathione mixed disulfides in cultured cells

“…In the present work, we provide, for the first time, a 3‐D image of the intracellular distribution of GSH, by combining the specificity of polyclonal antibody labeling for GSH with the ability of confocal microscopy to provide sectional images from stained cells. This represents a development of the previously documented use of the antibody to assay GSH immunohistochemically (11, 12). Figure 1 details the staining pattern obtained by fluorescence microscopy of several cell types.…”

“…In view of the difficulties in using existing cytochemical methods to study intracellular GSH compartmentalization, we have explored the possibility of developing an immunocytochemical method, based on labeling fixed cell preparations with a GSH‐specific, polyclonal antibody that has hitherto been largely applied to histochemical analyzes (11, 12). Thus, in the present work, we report on the cellular localization of GSH in intact cells, using a combination of secondary fluorescence staining and confocal microscopy.…”

Role of the nervous system in sarcopenia and muscle atrophy with aging: strength training as a countermeasure

“…As suggested by Edström et al (2007) the loss of synaptic input in terms of stripping of afferent boutons from spinal MNs seems to be highly selective and is associated with signs of neuroaxonal degeneration. An effect of reactive oxygen species have been implicated as axons and axon terminals with signs of neurodegeneration and/or neuroaxonal dystrophy have been observed to contain increased levels of total glutathione indicating redox stress (Ramirez‐Leon et al, 1999). Further, it has been suggested that axon impairment during aging may start as a distal process (cf.…”