The design of the biological experiments, with three treatments groups—healthy controls, a CFA group with animals subjected to peripheral inflammation only and a full EAE treatment, allowed for separation of the concentration changes caused by EAE from possible variations due to adjuvant injection, including variations due to animal growth or peripheral inflammation.
Several significant changes in concentrations of metabolites both at the onset and on the peak of the disease were detected. Dissimilar patterns of changes observed on both time points may suggest activation of different metabolic pathways in the early and late stage of the disease. The early-stage EAE samples, taken on day 10 post inoculation resemble the moment of maximal CNS infiltration by blood monocytes and T-cells, however, at this moment no negative signs on health status are observed and animals do not have any neurological scores. Metabolite level changes in the early stage of the disease may be caused by processes related to the development of EAE and disruption of the blood-CSF barrier.
The full-stage EAE samples are taken on day 14, at the peak of the disease, when the paralysis of the animals is most severe, which is expressed in maximum neurological scores (see Fig. ). The EAE model employed in this study involves spontaneous recovery of animals. In fact, due to the slightly heterogeneous course of the disease, 2 out of 15 animals from the EAE14
group had neurological scores which had already decreased relative to the previous day. Therefore, one can assume that some metabolites observed as significantly regulated in this sample series might be connected to pathological processes involved in EAE progression or recovery. The observed weight loss of EAE14
animals poses a difficult challenge in interpretation of observed changes. It was shown in the literature that weight loss and dietary restriction may cause changes in metabolite profiles in body fluids (Bollard et al. 2005
). However, it is unknown how these changes affect CSF metabolic profile. For that reason the brief interpretation of biological relevance of observed changes presented below is mostly limited to compounds involved in processes previously associated with MScl and EAE.
Several metabolites targeted in this study and found significantly up/down-regulated were previously associated with molecular processes interconnected with development of MScl and EAE. Glutamic acid (glutamate), glutamine, gamma-aminobutyric acid (GABA), asparagine and taurine are involved in excitotoxicity and energy metabolism. Arginine and citrulline play a key role in NO synthesis. Arginine, ornithine and putrescine are involved in polyamine synthesis. Alanine and branched-chain amino acids (valine, leucine, isoleucine) are involved in energy metabolism. See Table for overview.
Overview of most important biological processes interconnected with significantly regulated changes in metabolome detected in this study
-glutamic acid (glutamate) is a ubiquitous neurotransmitter with several identified receptors and transport proteins both in neural and glial cells (Danbolt 2001
). Over-stimulation of glutamate receptors may cause cell death due to excitotocity. Indeed, elevated levels of CSF glutamate were reported in clinical cases of multiple sclerosis (Sarchielli et al. 2003
). Contrary to previous reports, as shown in Table , the level of glutamic acid in this study shows a statistically significant decrease in concentration at the peak of the disease, while no significant change is observed at the early stage. However, the glutamine, which is closely interconnected with glutamate through glutamine-glutamate cycle in neurons and astrocytes, shows down-regulation at the onset and up-regulation with full stage EAE. Conversion of glutamate into glutamine by glutamine synthetase (GS) enzyme localized in astrocytes is a key process protecting neurons from ammonia toxicity (Norenberg and Martinez-Hernandez 1979
) and the only pathway of ammonia disposal due to the lack of complete urea cycle in CNS (Wiesinger 2007
). Available evidence shows up-regulation of GS in astrocytes present in demyelinating lesions as assessed in a study of post mortem brain tissue of MS patients (Newcombe et al. 2008
). However, a similar study performed in MBP-induced mouse EAE shows down-regulation of this enzyme (Hardin-Pouzet et al. 1997
At the peak of the disease we observe a significant gamma-aminobutyric acid (GABA) increase. GABA is synthesized from glutamate by glutamate decarboxylase. It was shown that GABA, an inhibitory neurotransmitter, may attenuate glutamate-induced excitotoxicity (Ohkuma et al. 1994
). In our study we observe significant increase in concentration of another inhibitory neurotransmitter, taurine, which was also shown to be protective against excitotoxicity (El Idrissi and Trenkner 1999
It should also be emphasized that glutamate, besides having a profound role as a neurotransmitter in CNS, is one of the key metabolites in energy metabolism of neurons, glia and immune cells (Newsholme et al. 1999
). It is possible that the global CSF levels of glutamine and glutamate involved in energy metabolism dominate over local high and excitotoxic concentrations in the most affected regions of brain and spinal cord. The observed discrepancy between available results on glutamate up- and down-regulation in EAE/MS suggests it may be involved in multiple different molecular processes associated with neuroinflammation and neurodegeneration.
Cytotoxicity interconnected with peroxynitrylation
Peroxynitrile anion (ONOO−
) is a very reactive oxidizing agent, capable of inducing cell death through multiple pathways (Szabó et al. 2007
). It emerges in the reaction between nitric oxide, produced mainly by inductive nitric oxide synthetase (iNOS) in activated immune cells and microglia, and the free radical superoxide O2−
, coming from oxidative phosphorylation cycle in mitochondria. Currently available evidence suggests, besides promotion of peroxynitrile synthesis, that NO plays a complex regulatory role in mediating the immune response (van der Veen et al. 1997
). Numerous reports suggest activity of peroxynitrile in active lesions in MScl (Smith and Lassmann 2002
), as well as in EAE (van der Veen et al. 1997
In our study we observe significant down-regulation of arginine, the main substrate for NO synthesis during the early stage of EAE. This effect may be interconnected with intense production of NO by cytotoxic T-cells, macrophages and activated microglia during development of neuroinflammation. However, the exact role of NO in EAE and MScl development remains unclear. Available reports show that inhibition of NO synthesis may attenuate (Ding et al. 1998
) or enhance (Zielasek et al. 1995
Interestingly, a second product of NO synthesis, citrulline, is also significantly down-regulated in the early stage of EAE, while one could expect increased production of citrulline linked to increased NO synthesis. However, immune and CNS cells are able to re-synthesize arginine in the so-called arginine-NO cycle (see Fig. , solid line), This cycle can be considered as a shortcut of the urea cycle (Fig. , dashed line), which due to the lack of ornithine carbamoyltransferase (OCT) is not complete in the CNS (Wiesinger 2001
). It was demonstrated that arginine deficiency does not inhibit NO synthesis by T-cells, which under such circumstances are able to increase uptake of citrulline and up-regulate enzymes responsible for arginine synthesis (Bansal et al. 2004
). Due to the involvement of arginine in other reactions, the citrulline–NO cycle does not operate in a stoichiometric manner, resulting in simultaneous decrease of arginine and citrulline levels.
Fig. 5 Overview of pathways of arginine metabolism in CNS. Hexagons depict metabolites and squares—reactions. Compounds measured in our study are marked in grey. Orinithine transcarbamoylase (OTC), argininosuccinate synthetase (ASS), argininosuccinate (more ...)
It should be also noted that the most significant alternative reaction of arginine utilization is the synthesis of ornithine by the enzyme arginase (ARG). This reaction is known to take part in regulating NO synthesis by reducing the amount of available arginine (Bansal and Ochoa 2003
). In the early stage of EAE we observe a decrease in the concentration of ornithine, supporting the hypothesis that arginine is probably being utilized by NO synthesis.
During the peak of EAE, arginine levels do not differ from controls, while citrulline levels continue to decrease. This may be explained by the reduction of NO synthesis interconnected with continued activity of other citrulline-NO cycle enzymes. At the same time we observe up-regulation of products of an alternative pathway of arginine utilization. The ornithine levels return to their normal states after significant decreases during the EAE onset, and this correlates with the significant 20-fold increase of putrescine levels, synthesised from ornithine by ornithine decarboxylase (see Fig. ). These results may suggest a switch in arginine metabolism from nitric oxide production towards the synthesis of ornithine and further to polyamine synthesis, which could be associated with compensatory anti-inflammatory response and recovery. Interestingly, the rat with the highest observed level of putrescine is a clear outlier from the rest of the group (animal 76 on Fig. ) and is one of two animals in the group that showed a neurological score decrease on the last day of experiment.
Putrescine was reported to be up-regulated in EAE (Bolton and Paul 2006
), however this change was associated with disease progression rather than recovery mechanisms. On the other hand, the beneficial effect of increased polyamine synthesis on post-injury recovery of CNS was demonstrated in literature (Gilad et al. 1996
; Cai et al. 2002
). Available reports show the potential of putrescine to block NMDA glutamate receptors (Williams 1997
) and therefore could possibly attenuate excitotoxic effects of glutamate.
It was postulated previously that over the course of inflammation, metabolism of arginine switches from NO synthesis in the early stage to arginine utilization in polyamine synthesis during tissue repair and wound healing (Nelin et al. 2007
Protection from oxidative stress
It was shown that antioxidants, such as uric acid, inhibit inflammation in EAE (Hooper et al. 2000
). The inhibitory neurotransmitter taurine, observed as up-regulated in our study, is also known for its antioxidant and anti-inflammatory activity (Li et al. 2007
). Levels of taurine increase over the course of EAE, suggesting that they may play a role in protection and recovery mechanisms. As the neurotoxicity of peroxynitrile might be dependent on the availability of superoxide rather than nitric oxide alone, it was suggested that MScl may be in fact connected with a malfunction of mitochondria (Mao and Reddy 2010
) or imbalance of ATP synthesis and energy metabolism (Cadoux-Hudson et al. 1991
; Amorini et al. 2009
Besides changes discussed above, we also observe other changes in the metabolome that cannot be clearly connected to already established hypotheses of MScl/EAE mechanisms. In the early stage of the EAE we observe decreased levels of alanine and branched-chain amino-acids (BCAA: leucine, isoleucine and valine). Alanine and BCAAs are known to be utilized as a source for pyruvate for energy metabolism or de novo synthesis of macromolecules within neural (Hutson et al. 2007
) and immune cells (Li et al. 2007
). As the onset of EAE is associated with maximum infiltration of the CNS by blood monocytes and T-cells, the observed decrease of BCAAs and alanine may suggest these metabolites are utilized for energy metabolism by invading cells. Alternatively, pro-inflammatory cytokines released during EAE development may cause changes in energy metabolism of native CNS cells.
In the full stage EAE, among other changes, we observe a significant increase of O
-phosphoethanolamine concentration, which is an important intermediate in phospholipid metabolism. Wheeler et al. (2008
) reported changes in lipid contents in normal appearing white and grey matter of MScl patients, suggesting changes in lipid metabolism including increased sphingolipid turnover and phospholipid synthesis. The detected increase of O
-phosphoethanolamine might be interconnected with these processes.