GLS hyperactivity causes glutamate excess, infantile cataract and profound developmental delay

Abstract

Loss-of-function mutations in glutaminase (GLS), the enzyme converting glutamine into glutamate, and the counteracting enzyme glutamine synthetase (GS) cause disturbed glutamate homeostasis and severe neonatal encephalopathy. We report a de novo Ser482Cys gain-of-function variant in GLS encoding glutaminase associated with profound developmental delay and infantile cataract. Functional analysis demonstrated that this variant causes hyperactivity and compensatory downregulation of GLS expression combined with upregulation of the counteracting enzyme GS, supporting pathogenicity. Ser482Cys-GLS likely improves the electrostatic environment of the GLS catalytic site, thereby intrinsically inducing hyperactivity. Alignment of +/-12.000 GLS protein sequences from >1000 genera, revealed extreme conservation of Ser482, to the same degree as catalytic residues. Together with the hyperactivity, this indicates that Ser482 is evolutionarily preserved to achieve optimal -but submaximal- GLS activity. In line with GLS hyperactivity, increased glutamate and decreased glutamine concentrations were measured in urine and fibroblasts. In the brain (both grey and white matter), glutamate was also extremely high and glutamine almost undetectable, using ultra-high field magnetic resonance spectroscopic imaging. Considering the neurotoxicity of glutamate when present in excess, the strikingly high glutamate concentrations measured in the brain provide an explanation for the developmental delay. Cataract, a known consequence of oxidative stress, was evoked in zebrafish expressing the hypermorphic Ser482Cys-GLS and could be alleviated by inhibition of GLS. The capacity to detoxify reactive oxygen species was reduced upon Ser482Cys-GLS expression, providing an explanation for cataract formation. In conclusion, we describe an inborn error of glutamate metabolism caused by a GLS hyperactivity variant, illustrating the importance of balanced GLS activity.

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“…Neurological disorders: Neuronal PAS domain-containing protein 2 (ASSRSS), which is highly polymorphic in autism spectrum disorder patients [ 60 , 61 ]; Circadian locomotor output cycles protein kaput (ASSRSS) relates to bipolar disorder [ 62 ]; Adenosine receptor A1 (VLPPLL), where sleep is significantly attenuated by the loss of adenosine A1 receptor expression [ 63 ]; BDNF/NT-3 growth factors receptor (SANLAA), alterations may cause temporal lobe epilepsy, memory impairment, anorexia nervosa, bulimia, Alzheimer’s disease [ 64 , 65 , 66 ]; Calcium/calmodulin-dependent protein kinase kinase 2 (PSLATV) is linked to disturbances of higher cognitive functions, such as working memory and executive function. as well as schizophrenia [ 67 ]; Endoplasmic reticulum mannosyl-oligosaccharide 1,2-alpha-mannosidase (LAFLLF) can cause mental retardation [ 68 ]; Glutaminase kidney isoform, mitochondrial (LQELGK) can associate with epileptic encephalopathy, infantile cataract, skin abnormalities leukocytoclasia at the surface of the dermis, focal vacuolar alterations, hyperkeratosis, parakeratosis, glutamate excess, and impaired intellectual development, global developmental delay, progressive ataxia, and elevated glutamine [ 69 , 70 , 71 ]; Mitochondrial glutamate carrier 1 (RLQSLQ) relates to neonatal myoclonic epilepsy [ 72 ]. …”

Inborn errors of enzymes in glutamate metabolism

“…A de novo hypermorphic GLS variant has recently been reported in a single patient affected with cataract, profound developmental delay, axial hypotonia and agitated, selfinjuring behaviour. 19 Glutamate and glutamine concentrations in plasma and CSF from the patient were within the normal range. In urine and in brain, as detected by in vivo magnetic resonance spectroscopic imaging (MRSI), glutamate concentrations were increased and glutamine concentrations decreased, in line with the tissue-specific expression of GLS.…”

“…In GS deficiency, glutamine deficiency results in reduced NAD + synthesis, which plays an important role in redox reactions . In GLS hyperactivity, glutamate excess is postulated to decrease redox buffer capacity . As oxidative stress is associated with cataract, neurodegenerative disorders and epilepsy, this is a likely key player in the pathophysiology of these inborn errors of glutamate metabolism …”

“…Interestingly, in GLS hyperactivity, glutamate and glutamine levels are normal in plasma and CSF, while both are deviant when analysed using brain MRS and in urine in line with tissue specific expression of GLS. 19 The discrepancy between CSF and brain MRS might be explained by the degree to which glutamate and glutamine levels are controlled by GLS. CSF is formed out of plasma by the plexus choroid, which consists of glial cells in which glutamate and glutamine levels are regulated by GS rather than GLS.…”

“…6 In GLS hyperactivity, glutamate excess is postulated to decrease redox buffer capacity. 19 As oxidative stress is associated with cataract, neurodegenerative disorders and epilepsy, this is a likely key player in the pathophysiology of these inborn errors of glutamate metabolism. 10,20,88 In UCDs, glutamine concentrations are increased as a consequence of hyperammonemia.…”