Modelling the impact of nitric oxide in neurodevelopment with an inherited metabolic disease of nitr

Modelling the impact of nitric oxide in neurodevelopment with an inherited metabolic disease of nitric oxide deficiency, argininosuccinate lyase deficiency.

Background: Ubiquitously expressed argininosuccinate lyase (ASL) is the only enzyme in mammals enabling the final step of arginine synthesis. ASL deficiency causes life-threatening hyperammonaemic episodes but patients develop developmental delay and epilepsy despite normal ammonia levels (1). Neuronal oxidative stress persists despite correction of ureagenesis (2). ASL enables the synthesis of central catecholamines by regulating tyrosine hydroxylase (TH) via nitrosylation in dopaminergic neurons of various midbrain basal ganglia such as locus coereleus (3), substantia nigra compacta and ventral tegmental area (4). Reduction of dopamine metabolites in ASL deficient mice causes ataxia and memory loss which can be corrected by nitric oxide donors (3, 4). ASL deficiency and subsequent TH downregulation causes tyrosine accumulation, which generates tyrosine aggregates leading to production of alpha-synuclein and supporting the “seeding theory” of neurodegeneration (4). This project explores the link between monogenic metabolic disorders, nitric oxide mediated impaired neurodevelopment and potentially neurodegeneration.

Aims/Objectives:
1. Develop midbrain dopaminergic (mDA) neurons from ASL-deficient human induced pluripotent stem cell (hiPSC) lines 
2. Study the pathophysiological mechanisms leading to neurodegeneration
3. Test novel therapies targeting the central nervous system: nitric oxide donors, mRNA and lentivirus mediated gene therapies.

Methods:
1. Available ASL-deficient hiPSC lines will be used to generate mDA neurons with an established protocol of Dr Barral (5). This will include cell (cell culture, immunostaining) and molecular biology (cDNA and RNA isolation, quantitative PCR, bulk transcriptomics).
2. Neurotransmitter (dopamine and GABA metabolites) measurement will be performed in Prof Heales and Dr Eaton’s laboratory by high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GCMS), respectively.
3. Cell-based assays will assess the presence of neurodegenerative and oxidative stress     markers, mitochondrial and autophagy dysfunction by immunostaining, western blot and qPCR. The more relevant assays will be used to test the efficacy of novel therapies generated in-house or obtained via ongoing collaboration with pharmaceutical companies.
4. Transcriptomic analysis will enable learning skills in data sciences. 
Consumables will be funded by Dr Baruteau’s MRC Clinician Scientist Fellowship. 

Timeline (if applicable):
Aim 1: Establish patient-derived mDA neuronal model: Month 0-9 (9 months)
Aim 2: Pathophysiology of neurodegenerative features: Month 10-24 (15 months)
Aim 3: Assessment of novel therapies: Month 24-36 (12 months)

References:

1. Baruteau, J., et al., Expanding the phenotype in argininosuccinic aciduria: need for new therapies. J Inherit Metab Dis, 2017. 40(3): p. 357-368.

2. Baruteau, J., et al., Argininosuccinic aciduria fosters neuronal nitrosative stress reversed by Asl gene transfer. Nat Commun, 2018. 9(1): p. 3505.

3. Lerner, S., et al., ASL Metabolically Regulates Tyrosine Hydroxylase in the Nucleus Locus Coeruleus. Cell Rep, 2019. 29(8): p. 2144-2153 e7.

4. Lerner, S., et al., Tyrosine accumulation following ASL loss promotes neurodegeneration. Submitted. 5. Ng, Barral et al., Gene therapy restores dopamine transporter expression and ameliorates pathology in iPSC and mouse models of infantile parkinsonism. Sci. Transl. Med. 13, eaaw1564. 2021.