Biochemical Studies in Encephalomyocarditis (EMS) of pigs

The topic of the project is to investigate whether compounds of the kynurenine pathway of tryptophan metabolism within the central nervous system e.g. brain and spinal cord and peripheral system e.g. heart may play a role in the formation of behavioural and neuropathological signs of experimentally or naturally EMCV infected piglets. EMCV is stated as an infection of heart tissue, spinal cord and brain caused by a group of Cardiovirus (Picornaviridae). Rodents, squirrels, racoons, monkeys, elephants are a major source of the infection. For humans there are no indications for infection, however a possibility can not be excluded in immune compromised persons. Only in young pigs the virus causes a sporadic disease characterized primarily by sudden death. Early clinical signs may include anorexia, depression, and breathing difficulties. A notable susceptibility of young but not old rat brains to EMCV and an accompanied neuronal degeneration in the hippocampus in vitro has been demonstrated. It has been also suggested that the predominant target cells for EMCV persistence are macrophages. There is abundant evidence that the tryptophan metabolism is up-regulated in certain cells during inflammatory states, e.g. in activated macrophages and microglial cells producing toxic tryptophan derivatives in addition to other cytotoxines. Viral infection attributes to induction of indoleamine-2,3-dioxygenase, the first enzyme of the kynurenine pathway which converts L-tryptophan to L-kynurenine. L-kynurenine metabolites, thus 3-hydroxy- kynurenine and quinolinic acid have potent neurotoxic properties, while kynurenic acid, a closely related metabolite, displays opposite activity and the disturbance of their synthesis within the central nervous system and/or in the periphery might underlie the neuropathological phenomena of EMCV infected piglets. Recently we have found that kynurenic acid influences significantly the heart mitochondrial function and this finding could be of exceedingly importance in the understanding of EMVC induced cardiac and CNS impairment. The following questions will be investigated in this project: 1. Is there a correlation between the alteration of content of L-kynurenine, kynurenic acid and 3-OH-kynurenine in the CSF and serum and the severity of neurological impairment in piglets infected with EMCV compared to controls? 2. Is there a relation of postulated alterations of L-kynurenine, kynurenic acid and 3-OH-kynurenine levels e.g. to enzyme activities changes, which are responsible for the kynurenic acid formation (kynurenine aminotransferase I and kynurenine aminotransferase II) in different regions of CNS and in the heart? 3. Are there organ specific (heart vs. brain and spinal cord) alterations of kynurenic acid metabolism and changes of mitochondrial Complex I activity in EMCV piglets as compared to controls? Subsequently correlations between activities of neuronal markers of GABAergic and cholinergic systems and kynurenine aminotransferases activities in the CNS will be elaborated. This study should clarify the significances of kynurenine metabolism with respect to formation of behavioural and neuropathological signs within the central nervous system e.g. brain and spinal cord and peripheral system e.g. heart of experimentally or naturally EMCV infected piglets. Futhermore this study will provide for the first time a proof that elevation of kynurenic acid metabolism is involved in the impairment of Complex I mitochondrial machinery. The proof of our in vitro findings concerning the effect of kynurenic acid on the mitochondria could be demonstrated with the study of EMCV infected piglets and could have a notable clinical impact with respect to therapeutical strategies and would have a pivotal theoretical impact with respect to understanding of aging and related processes. This study has been elaborated as a cooperation with an ongoing EU-Project Encephalomyocarditis by piglets FAIR6-CT98-4146 (a. Univ.-Prof. Dr. N. Nowotny, Clinical Virology, VU Vienna). Encephalomyocarditis virus (EMCV) is classified within the genus Cardiovirus of the family Picornaviridae; as it is already indicated in the name of the virus, EMCV mainly affects heart tissue and the central nervous system Clinically, EMCV causes a sudden death syndrome in piglets (due to acute myocarditis), and in adults the infection may only lead to reproductive failure. The dysfunction of heart has been suggested to be involved, however the mechanism(s) has(ve) not been elaborated yet. In addition, the viral infection is accompanied by alterations in the central nervous system, but no reliable data are available. A notable susceptibility of young but not old brain to EMCV and accompanied neuronal degeneration in the hippocampus in vitro has been demonstrated recently. L- kynurenine is a major intermediate in the oxidative metabolism of L-tryptophan leading to niacin formation. In the cell, L-kynurenine undergoes transamination to form kynurenic acid with a neuroprotectine activity or hydroxylation followed by transamination and oxygenation to produce 3-hydroxy kynurenine and quinolinic acid with neurotoxic activities. The increase of tryptophan metabolism in the peripheral and central nervous system in human and non-human subjects with inflammatory diseases (viral, bacterial, fungal and parasitic infections, autoimmune diseases, meningitis and septicaemia) have been attributed to induction of indoleamine-2,3- dioxygenase the first enzyme of the kynurenine pathway which converts L-tryptophan to L-kynurenine. Our recently revealed data indicated that kynurenic acid influences significantly heart mitochondrial function by increasing oxygen consumption and lowering ATP synthesis. Furthermore our data also indicated that complex I of the heart mitochondrial respiratory chain involving NADH:ubiquinone-oxidoreductase is more sensitive to the alteration of KYNA metabolism. Since the synthesis of KYNA from L-KYN takes a place in the mitochondria therefore a deterioration of the respiratory parameters by KYNA may be of particular importance. No effect on heart mitochondria function has been observed by quinolinic acid or 3-OH-kynurenine. Beside the known neuroprotective capacities of kynurenic acid its ability to influence mitochondria function represent significant finding which need to be elaborated and confirmed by further investigation. Infection in piglets is accompanied by neurodegeneration in the brain and spinal cord as has been show by histopathological investigation. Since there is substantial evidence that several metabolites of the kynurenine pathway are involved in inflammatory neurological disorders it seems possible that the formation of symptoms in EMC piglets is associated with changes in kynurenine metabolism. To data, no study has investigated this hypothesis in human or animal model. Already, our preliminary work revealed an abnormal elevation of KYNA levels in serum of EMCV piglets (Baran observation, no published data) and this data strongly suggest that changes of KYNA metabolism in EMCV could be important in respect to bioenergetic mitochondrial machinery in heart and in the brain function, respectively. Since an alteration of GABAergic and cholinergic neurons due to excitotoxic events (immune activation and also inflammatory processes) has been well documented in animal experimental studies (Saito 1991; Stone 1993 see, Ref.; Baran et al., 1987, Baran et al., 1994; Pitt, 2000) therefore the measurement of this neuronal markers in brain regions will provide also significant indication for neurodegenerative processes in the CNS of EMVC piglets.

The aim of the project "Biochemical Studies in Encephalomyocarditis (EMC) of pigs" was to investigate whether compounds of the kynurenine pathway of tryptophan metabolism within the central nervous system and peripheral system e.g. heart may play a role in the formation of behavioural and neuropathological signs of experimentally or naturally EMCV infected piglets. EMCV is stated as an infection of heart tissue, spinal cord and brain caused by a group of Cardiovirus (Picorna viridae). Rodents, squirrels, racoons, monkeys, elephants are a major source of the infection. For humans there are no indications for infection, however a possibility can not be excluded in immune compromised persons. Only in young pigs the virus causes a sporadic disease characterised primarily by sudden death. Early clinical signs may include anorexia, depression, and breathing difficulties. A notable susceptibility of young rat brains to EMCV and an accompanied neuronal degeneration in the hippocampus in in vitro studies has been demonstrated. It has been also suggested that the predominant target cells for EMCV persistence are macrophages. There is abundant evidence that the tryptophan metabolism is up-regulated in certain cells during inflammatory states, e.g. in activated macrophages and microglial cells producing toxic tryptophan derivatives in addition to other cytotoxines. Viral infection attributes to induction of indoleamine-2,3-dioxygenase, the first enzyme of the kynurenine pathway which converts L-tryptophan to L-kynurenine. L-kynurenine metabolites, thus 3-hydroxy- kynurenine and quinolinic acid have potent neurotoxic properties, while kynurenic acid, a closely related metabolite, displays opposite activity and the disturbance of their synthesis within the central nervous system and/or in the periphery might underlie the neuropathological phenomena of EMCV infected piglets. Recently we have found that kynurenic acid influences significantly the heart mitochondrial function and this finding could be of exceedingly importance in the understanding of EMVC induced cardiac and CNS impairment. Correlation between increased kynurenic acid levels and ß-microglobulin in the serum and the severity of neurological impairment in piglets infected with EMCV represents significant findings. With respect to our in vitro findings concerning the effect of kynurenic acid on the mitochondria function a reduction of oxygen consumption in brain and heart homogenate of EMCV infected piglets could be observed. We found in contrary to other species (rat or human) that in peripheral organs of pigs and piglets the kynurenine metabolism shows a very low activity - indicating a species specific difference. The alterations of markers of excitatory i.e. cholinergic and inhibitory i.e. GABAergic systems in the brain regions after EMCV infection have been observed and this finding could suggest the induction of neurodegenerative processes due to EMCV infection.