Final Report Summary - NO-BRAINER (Developing therapies for Nitric Oxide (NO) related neurodegeneration)
Argininosuccinic aciduria is a urea cycle disorder caused by mutations in the enzyme
Argininosuccinic lyase. For unknown reason, in spite of early diagnosis and initiation of therapy and even in the absence of documented hyperammonemia, patients with ASL deficiency (ASLD), have a higher incidence of hypertension and neurocognitive abnormalities as compared to subjects with other urea cycle disorders. Outside the liver, ASL is the only enzyme able to generate endogenous arginine. Although arginine as a substrate has multiple metabolic fates, undoubtedly its most studied product is nitric oxide, (NO). The current paradigm of how NO production is regulated focuses at the level of nitric oxide synthase (NOS), with respect to substrate and co-factor availability and the precise spatial and temporal arrangement of protein complexes. However, the respective unique or combined genetic deficiencies of the NOS isoforms exhibit relatively modest phenotypes in mice. Moreover, approaches targeted at modulating NOS activities have not successfully translated into therapy. All NOS isoforms are dependent on arginine as their sole substrate for NO synthesis and interestingly, only one enzyme in mammals- argininosuccinic lyase (ASL) can generate endogenous arginine.
We found that ASL is structurally essential for the utilization of arginine for NO synthesis. Encouragingly, treating ASLD patient with NO supplements indeed improved his neurocognitive function. The neurocognitive delays in ASLD together with the improvement in neurocognitive function following treatment with NO donors, suggest that ASL and NO have a role in neuronal function at homeostasis. We thus hypothesized that generating mice with specific deletion of Asl at different brain regions, will allow us to dissect the role of Asl/ NO in cognitive function at normal and pathological CNS conditions, for therapeutic applications.
To pursue my hypothesis, we used a multidisciplinary approach, combining system and cell-biology methodologies in vitro and in vivo. First, to dissect the importance of Asl/NO in neuronal function, we assayed the expression of Asl in wild type mice brains. We found that Asl is expressed in several specific brain regions which are responsible for brain metabolism, cognitive functions and regulation of the sympathetic activity. We next started breeding our novel Asl conditional mouse model with brain specific Cre’s to achieve brain region specific Asl knockouts. We have by now generated three mouse models with Asl deletion specific to the forebrain (Aslf/f;CampKCre+/-), dopaminergic cells (Aslf/f;THCre+/-) and neuronal specific (Aslf/f;NestinCre+/-). Our results show that Asl deletion in specific brain regions which relate to the sympathetic system, leads to deficiency in NO which is manifested clinically as hypertension and as an inappropriate stress response.
Thus, our results support that we have recapitulated the human CNS related phenotypes of ASLD disease in a tissue specific manner in mice and suggest central metabolic regulation of the sympathetic system. Thus, our continuing research is focusing on the regulatory role NO plays in the sympathetic system. We believe such findings will have clinical implications for treatment of ASLD patients and might improve their quality of life and cognitive abilities. More broadly, our results are expected to shed light on the role of ASL and NO in neuro behavior, cognition and sympathetic system regulation.
Argininosuccinic lyase. For unknown reason, in spite of early diagnosis and initiation of therapy and even in the absence of documented hyperammonemia, patients with ASL deficiency (ASLD), have a higher incidence of hypertension and neurocognitive abnormalities as compared to subjects with other urea cycle disorders. Outside the liver, ASL is the only enzyme able to generate endogenous arginine. Although arginine as a substrate has multiple metabolic fates, undoubtedly its most studied product is nitric oxide, (NO). The current paradigm of how NO production is regulated focuses at the level of nitric oxide synthase (NOS), with respect to substrate and co-factor availability and the precise spatial and temporal arrangement of protein complexes. However, the respective unique or combined genetic deficiencies of the NOS isoforms exhibit relatively modest phenotypes in mice. Moreover, approaches targeted at modulating NOS activities have not successfully translated into therapy. All NOS isoforms are dependent on arginine as their sole substrate for NO synthesis and interestingly, only one enzyme in mammals- argininosuccinic lyase (ASL) can generate endogenous arginine.
We found that ASL is structurally essential for the utilization of arginine for NO synthesis. Encouragingly, treating ASLD patient with NO supplements indeed improved his neurocognitive function. The neurocognitive delays in ASLD together with the improvement in neurocognitive function following treatment with NO donors, suggest that ASL and NO have a role in neuronal function at homeostasis. We thus hypothesized that generating mice with specific deletion of Asl at different brain regions, will allow us to dissect the role of Asl/ NO in cognitive function at normal and pathological CNS conditions, for therapeutic applications.
To pursue my hypothesis, we used a multidisciplinary approach, combining system and cell-biology methodologies in vitro and in vivo. First, to dissect the importance of Asl/NO in neuronal function, we assayed the expression of Asl in wild type mice brains. We found that Asl is expressed in several specific brain regions which are responsible for brain metabolism, cognitive functions and regulation of the sympathetic activity. We next started breeding our novel Asl conditional mouse model with brain specific Cre’s to achieve brain region specific Asl knockouts. We have by now generated three mouse models with Asl deletion specific to the forebrain (Aslf/f;CampKCre+/-), dopaminergic cells (Aslf/f;THCre+/-) and neuronal specific (Aslf/f;NestinCre+/-). Our results show that Asl deletion in specific brain regions which relate to the sympathetic system, leads to deficiency in NO which is manifested clinically as hypertension and as an inappropriate stress response.
Thus, our results support that we have recapitulated the human CNS related phenotypes of ASLD disease in a tissue specific manner in mice and suggest central metabolic regulation of the sympathetic system. Thus, our continuing research is focusing on the regulatory role NO plays in the sympathetic system. We believe such findings will have clinical implications for treatment of ASLD patients and might improve their quality of life and cognitive abilities. More broadly, our results are expected to shed light on the role of ASL and NO in neuro behavior, cognition and sympathetic system regulation.