Abstract
The cerebral cortex is the region of the mammalian brain responsible for controlling higher cognitive functions. Metabolic processes in the brain are unique, with the metabolic interplay between neuronal and glial cells vital for cerebral function. Neurons rely on metabolic support from glial cells through both bioenergetic and biosynthetic pathways. Despite the understanding of metabolomics in the mature cerebral cortex, the role of metabolic pathways in neurodevelopment is an emerging field. The study of inborn errors of metabolism, that have a neurological component to their pathology, informs on biological pathways that contribute to neurological function. As inborn errors of metabolism are phenotypically heterogeneous, genetic and biochemical analyses have greatly aided the identification and diagnosis of genetic metabolic disorders.
The focus of this thesis is a rare, novel disorder of glutamine metabolism. Glutamine synthetase, the enzyme that synthesizes glutamine endogenously, is pivotal for the generation of neurotransmitters glutamate and gamma-aminobutyric acid and is the primary mechanism of ammonia detoxification in the mature brain. A rare loss-of-function disorder, glutamine synthetase deficiency disorder, has been described previously in the literature. Affected individuals presented with neurological defects and abnormal biochemical profiles. An allelic disorder functioning through a stabilization mechanism is described in this work.
Nine individuals with severe developmental delay, seizures, and white matter abnormalities but normal plasma and cerebrospinal fluid biochemistry were ascertained. These individuals had de novo heterozygous variants clustered at the 5’ of GLUL, encoding glutamine synthetase. Seven of these individuals had variants within the canonical start codon, c.3G>A, c.1A>T, c.1A>C and c.1A>G with the c.1A>G variant being recurrent in this cohort. Two individuals had variants located on the splice boundary of a 5’UTR exon, c.-13-2A.G and c.-13-1G>A, which were found to disrupt 5’UTR splicing resulting in splice exclusion of the initiation codon.
In vitro analyses were performed to determine the protein effect of start-loss GLUL variants. When the canonical translation initiation site was disrupted, either directly or via splice exclusion, a downstream AUG codon was utilized resulting in a truncated protein isoform lacking 17 amino acids from the N-terminal. Within the truncated region is a N-terminal degron, that post-translationally regulates GS levels by enabling the ubiquitin-mediated degradation of GS in a glutamine-induced manner. Transfection-based experiments determined that truncated start-loss GS is stable, enzymatically competent and able to interact with full-length subunits but insensitive to negative feedback by glutamine. Consequently, it was proposed to call this rare disorder glutamine synthetase stabilization disorder. Analysis of human single-cell transcriptomes demonstrated that GLUL is widely expressed in neuro- and glial-progenitor cells and mature astrocytes but not in post-mitotic neurons.
The two allelic disorders, with distinct aetiologies, underlines the importance of tight regulation of glutamine metabolism during neurodevelopment in humans and expands the spectrum of clinical presentations associated with the GLUL locus. This newfound understanding of GS dysregulation, specifically the stabilisation of GS and its effects in the context of a neurodevelopmental disorder, is significant in advancing the current understanding of the role of glutamine metabolism in cortical development and function. While promising, this work also identifies areas of future work to further understand the neurological consequence of GS stabilisation, to not only enlighten processes operative in typical development but also inform on clinical management of individuals with start-loss GLUL variants.