NGLY1 Research Articles

Systemic gene therapy corrects the neurological phenotype in a mouse model of NGLY1 deficiency.

The cytoplasmic peptide:N-glycanase (NGLY1) is ubiquitously expressed and functions as a de-N-glycosylating enzyme that degrades misfolded N-glycosylated proteins. NGLY1 deficiency due to biallelic loss-of-function NGLY1 variants is an ultrarare autosomal recessive deglycosylation disorder with multisystemic involvement; the neurological manifestations represent the main disease burden. Currently, there is no treatment for this disease. To develop a gene therapy, we first characterized a tamoxifen-inducible Ngly1-knockout (iNgly1) C57BL/6J mouse model, which exhibited symptoms recapitulating human disease, including elevation of the biomarker GlcNAc-Asn, motor deficits, kyphosis, Purkinje cell loss, and gait abnormalities. We packaged a codon-optimized human NGLY1 transgene cassette into 2 adeno-associated virus (AAV) capsids, AAV9 and AAV.PHPeB. Systemic administration of the AAV.PHPeB vector to symptomatic iNgly1 mice corrected multiple disease features at 8 weeks after treatment. Furthermore, another cohort of AAV.PHPeB-treated iNgly1 mice were monitored over a year and showed near-complete normalization of the neurological aspects of the disease phenotype, demonstrating the durability of gene therapy. Our data suggested that brain-directed NGLY1 gene replacement via systemic delivery is a promising therapeutic strategy for NGLY1 deficiency. Although the superior CNS tropism of AAV.PHPeB vector does not translate to primates, emerging AAV capsids with enhanced primate CNS tropism will enable future translational studies.

Development of new NGLY1 assay systems - toward developing an early screening method for NGLY1 deficiency.

Cytosolic peptide: N-glycanase (PNGase/NGLY1 in mammals) is an amidase (EC:3.5.1.52) widely conserved in eukaryotes. It catalyzes the removal of N-glycans on glycoproteins, converting N-glycosylated Asn into Asp residues. This enzyme also plays a role in the quality control system for nascent glycoproteins. Since the identification of a patient with an autosomal recessive genetic disorder caused by NGLY1 gene dysfunction, known as NGLY1 deficiency or NGLY1 congenital disorder of deglycosylation (OMIM: 615273), in 2012, more than 100 cases have been reported worldwide. NGLY1 deficiency is characterized by a wide array of symptoms, such as global mental delay, intellectual disability, abnormal electroencephalography findings, seizure, movement disorder, hypolacrima or alacrima, and liver dysfunction. Unfortunately, no effective therapeutic treatments for this disease have been established. However, administration of adeno-associated virus 9 (AAV9) vector harboring human NGLY1 gene to an NGLY1-deficient rat model (Ngly1-/- rat) by intracerebroventricular injection was found to drastically improve motor function defects. This observation indicated that early therapeutic intervention could alleviate various symptoms originating from central nervous system dysfunction in this disease. Therefore, there is a keen interest in the development of facile diagnostic methods for NGLY1 deficiency. This review summarizes the history of assay development for PNGase/NGLY1 activity, as well as the recent progress in the development of novel plate-based assay systems for NGLY1, and also discusses future perspectives.

Intranasal oxytocin suppresses seizure-like behaviors in a mouse model of NGLY1 deficiency

NGLY1 deficiency is a genetic disease caused by biallelic mutations of the Ngly1 gene. Although epileptic seizure is one of the most severe symptoms in patients with NGLY1 deficiency, preclinical studies have not been conducted due to the lack of animal models for epileptic seizures in NGLY1 deficiency. Here, we observed the behaviors of male and female Ngly1−/− mice by video monitoring and found that these mice exhibit spontaneous seizure-like behaviors. Gene expression analyses and enzyme immunoassay revealed significant decreases in oxytocin, a well-known neuropeptide, in the hypothalamus of Ngly1−/− mice. Seizure-like behaviors in Ngly1−/− mice were transiently suppressed by a single intranasal administration of oxytocin. These findings suggest the therapeutic potential of oxytocin for epileptic seizure in patients with NGLY1 deficiency and contribute to the clarification of the disease mechanism.

ELISA-based highly sensitive assay system for the detection of endogenous NGLY1 activity

Cytosolic peptide:N-glycanase (NGLY1, PNGase) is an enzyme that cleaves N-glycans from misfolded glycoproteins. In 2012, a human genetic disorder, NGLY1 deficiency, was first reported to be caused by mutations of the NGLY1 gene. Since then, there has been rapid progresses on NGLY1 biology, and gene therapy has been proposed as a promising therapeutic option for NGLY1 deficiency. While a plasma/urine biomarker has also been developed for this disease, detection of NGLY1 activity could be another viable option for early diagnosis of NGLY1 deficiency. Thus far, several in vitro and in cellulo NGLY1 assays have been reported, but those assay systems have several issues that must be addressed in order to develop an assay system compatible for routine clinical examination. Here, we show a facile, highly sensitive in vitro assay system that could be used to detect NGLY1 activity by utilizing its sequence editing function, i.e. conversion of glycosylated Asn into Asp, followed by a detection of newly generated epitope (HA)-tag by anti-HA antibody. Using this ELISA-based assay, we detected endogenous NGLY1 activity in as little as 2 μg of crude extract, which is the equivalent of 5 × 103 cells. Our system also detects NGLY1 activity from cells with compromised NGLY1 activity, such as iPS cells from patient samples. This assay system could be applied in future clinical examinations to achieve an early diagnosis of NGLY1 deficiency.

Development of a fluorescence and quencher-based FRET assay for detection of endogenous peptide:N-glycanase/NGLY1 activity

Cytosolic peptide:N-glycanase (PNGase/NGLY1 in mammals) catalyzes deglycosylation of N-glycans on glycoproteins. A genetic disorder caused by mutations in the NGLY1 gene leads to NGLY1 deficiency, with symptoms including motor deficits and neurological problems. Effective therapies have not been established, though a recent study used the administration of an adeno-associated viral vector expressing human NGLY1 to dramatically rescue motor functions in young Ngly1–/– rats. Thus, early therapeutic intervention may improve symptoms arising from central nervous system dysfunction, and assay methods for measuring NGLY1 activity in biological samples are critical for early diagnostics. In this study, we established an assay system for plate-based detection of endogenous NGLY1 activity using a FRET-based probe. Using this method, we revealed significant changes in NGLY1 activity in rat brains during aging. This novel assay offers reliable disease diagnostics and provides valuable insights into the regulation of PNGase/NGLY1 activity in diverse organisms under different stress conditions.

A commentary on 'Patient-derived gene and protein expression signatures of NGLY1 deficiency'.

The cytosolic peptide:N-glycanase (PNGase; NGLY1 in human and PNG1 in budding yeast) is a deglycosylating enzyme widely conserved in eukaryotes. Initially, functional importance of this enzyme remained unknown as the png1Δ mutant in yeast did not exhibit any significant phenotypes. However, the discovery of NGLY1 deficiency, a rare genetic disorder with biallelic mutations in NGLY1 gene, prompted an intensification of research that has resulted in uncovering the significance of NGLY1 as well as the proteins under its influence that are involved in numerous cellular processes. A recent report by Rauscher et al. (Patient-derived gene and protein expression signatures of NGLY1 deficiency. J. Biochem. 2022; 171: 187-199) presented a comprehensive summary of transcriptome/proteome analyses of various cell types derived from NGLY1-deficient patients. The authors also provide a web application called 'NGLY1 browser', which will allow researchers to have access to a wealth of information on gene and protein expression signature for patients with NGLY1 deficiency.

NGLY1: A fascinating, multifunctional molecule

NGLY1, a cytoplasmic de-N-glycosylating enzyme is well conserved among eukaryotes. This enzyme has attracted considerable attention after mutations on the NGLY1 gene were found to cause a rare genetic disorder called NGLY1 deficiency. Recent explosive progress in NGLY1 research has revealed multi-functional aspects of this protein.

Generation and characterization of NGLY1 patient-derived midbrain organoids.

NGLY1 deficiency is an ultra-rare, autosomal recessive genetic disease caused by mutations in the NGLY1 gene encoding N-glycanase one that removes N-linked glycan. Patients with pathogenic mutations in NGLY1 have complex clinical symptoms including global developmental delay, motor disorder and liver dysfunction. To better understand the disease pathogenesis and the neurological symptoms of the NGLY1 deficiency we generated and characterized midbrain organoids using patient-derived iPSCs from two patients with distinct disease-causing mutations-one homozygous for p. Q208X, the other compound heterozygous for p. L318P and p. R390P and CRISPR generated NGLY1 knockout iPSCs. We demonstrate that NGLY1 deficient midbrain organoids show altered neuronal development compared to one wild type (WT) organoid. Both neuronal (TUJ1) and astrocytic glial fibrillary acid protein markers were reduced in NGLY1 patient-derived midbrain organoids along with neurotransmitter GABA. Interestingly, staining for dopaminergic neuronal marker, tyrosine hydroxylase, revealed a significant reduction in patient iPSC derived organoids. These results provide a relevant NGLY1 disease model to investigate disease mechanisms and evaluate therapeutics for treatments of NGLY1 deficiency.

N-glycoproteomics reveals distinct glycosylation alterations in NGLY1-deficient patient-derived dermal fibroblasts.

Congenital disorders of glycosylation are genetic disorders that occur due to defects in protein and lipid glycosylation pathways. A deficiency of N-glycanase 1, encoded by the NGLY1 gene, results in a congenital disorder of deglycosylation. The NGLY1 enzyme is mainly involved in cleaving N-glycans from misfolded, retro-translocated glycoproteins in the cytosol from the endoplasmic reticulum before their proteasomal degradation or activation. Despite the essential role of NGLY1 in deglycosylation pathways, the exact consequences of NGLY1 deficiency on global cellular protein glycosylation have not yet been investigated. We undertook a multiplexed tandem mass tags-labeling-based quantitative glycoproteomics and proteomics analysis of fibroblasts from NGLY1-deficient individuals carrying different biallelic pathogenic variants in NGLY1. This quantitative mass spectrometric analysis detected 8041 proteins and defined a proteomic signature of differential expression across affected individuals and controls. Proteins that showed significant differential expression included phospholipid phosphatase 3, stromal cell-derived factor 1, collagen alpha-1 (IV) chain, hyaluronan and proteoglycan link protein 1, and thrombospondin-1. We further detected a total of 3255 N-glycopeptides derived from 550 glycosylation sites of 407 glycoproteins by multiplexed N-glycoproteomics. Several extracellular matrix glycoproteins and adhesion molecules showed altered abundance of N-glycopeptides. Overall, we observed distinct alterations in specific glycoproteins, but our data revealed no global accumulation of glycopeptides in the patient-derived fibroblasts, despite the genetic defect in NGLY1. Our findings highlight new molecular and system-level insights for understanding NGLY1-CDDG.

Ever-expanding NGLY1 biology.

The cytosolic peptide:N-glycanase (PNGase; NGLY1 in humans) is a deglycosylating enzyme that is widely conserved in eukaryotes. This enzyme is involved in the degradation of misfolded N-glycoproteins that are destined for proteasomal degradation in the cytosol, a process that is called endoplasmic reticulum-associated degradation. Although the physiological significance of NGLY1 remained unknown until recently, the discovery of NGLY1 deficiency, a human genetic disorder bearing mutations in the NGLY1 gene, has led to explosive research progress regarding the functional characterization of this enzyme. For example, it is now known that NGLY1 can also act as an 'editing enzyme' to convert N-glycosylated asparagine residues to aspartate residues, thus introducing negative charges into a core peptide and modulating the function of the target molecule. Diverse biological processes have also been found to be affected by compromised NGLY1 activity. In this special issue, recent research progress on the functional characterization of NGLY1 and its orthologues in worm/fly/rodents, assay methods/biomarkers useful for the development of therapeutics and the comprehensive transcriptome/proteome of NGLY1-KO cells as well as patient-derived cells are discussed.

Assay for the peptide:N-glycanase/NGLY1 and disease-specific biomarkers for diagnosing NGLY1 deficiency.

Cytosolic peptide:N-glycanase (NGLY1 in mammals), a highly conserved enzyme in eukaryotes, catalyses the deglycosylation of N-glycans that are attached to glycopeptide/glycoproteins. In 2012, an autosomal recessive disorder related to the NGLY1 gene, which was referred to as NGLY1 deficiency, was reported. Since then, more than 100 patients have been identified. Patients with this disease exhibit various symptoms, including various motor deficits and other neurological problems. Effective therapeutic treatments for this disease, however, have not been established. Most recently, it was demonstrated that the intracerebroventricular administration of an adeno-associated virus 9 vector expressing human NGLY1 during the weaning period allowed some motor functions to be recovered in Ngly1-/- rats. This observation led us to hypothesize that a therapeutic intervention for improving these motor deficits or other neurological symptoms found in the patients might be possible. To achieve this, it is critical to establish robust and facile methods for assaying NGLY1 activity in biological samples, for the early diagnosis and evaluation of the therapeutic efficacy for the treatment of NGLY1 deficiency. In this mini review, we summarize progress made in the development of various assay methods for NGLY1 activity, as well as a recent progress in the identification of NGLY1 deficiency-specific biomarkers.

Physiological importance of NGLY1, as revealed by rodent model analyses

Cytosolic peptide:N-glycanase (NGLY1) is an enzyme that cleaves N-glycans from glycoproteins that has been retrotranslocated from the endoplasmic reticulum (ER) lumen into the cytosol. It is known that NGLY1 is involved in the degradation of cytosolic glycans (non-lysosomal glycan degradation) as well as ER-associated degradation, a quality control system for newly synthesized glycoproteins. The discovery of NGLY1 deficiency, which is caused by mutations in the human NGLY1 gene and results in multisystemic symptoms, has attracted interest in the physiological functions of NGLY1 in mammals. Studies using various animal models led to the identification of possible factors that contribute to the pathogenesis of NGLY1 deficiency. In this review, we summarize phenotypic consequences that have been reported for various Ngly1-deficient rodent models and discuss future perspectives to provide more insights into the physiological functions of NGLY1.

Expanding DSD Phenotypes Associated with Variants in the DEAH-Box RNA Helicase DHX37

Missense variants in the RNA-helicase DHX37 are associated with either 46,XY gonadal dysgenesis or 46,XY testicular regression syndrome (TRS). DHX37 is required for ribosome biogenesis, and this subgroup of XY DSD is a new human ribosomopathy. In a cohort of 140 individuals with 46,XY DSD, we identified 7 children with either 46,XY complete gonadal dysgenesis or 46,XY TRS carrying rare or novel DHX37 variants. A novel p.R390H variant within the RecA1 domain was identified in a girl with complete gonadal dysgenesis. A paternally inherited p.R487H variant, previously associated with a recessive congenital developmental syndrome, was carried by a boy with a syndromic form of 46,XY DSD. His phenotype may be explained in part by a novel homozygous loss-of-function variant in the NGLY1 gene, which causes a congenital disorder of deglycosylation. Remarkably, a homozygous p.T477H variant was identified in a boy with TRS. His fertile father had unilateral testicular regression with typical male genital development. This expands the DSD phenotypes associated with DHX37. Structural analysis of all variants predicted deleterious effects on helicase function. Similar to all other known ribosomopathies, the mechanism of pathogenesis is unknown.

Congenital disorder of deglycosylation associated with N-glycanse 1 deficiency

Together with the lysosomal storage diseases, N-glycanase 1 deficiency is a congenital disorder of deglycosylation, which has been diagnosed in 27 patients, including two of them from Poland. The pathogenesis remains unknown, however, the main role is attributed to the disturbed endoplasmic reticulum-associated protein degradation process. The most characteristic symptoms include global developmental disability, hyperkinetic movement disorder, hypo-/alacrimia, and elevated serum transaminases. Identification of pathogenic variants in the NGLY1 gene is required to confirm the diagnosis.

NGLY1 deficiency: Novel patient, review of the literature and diagnostic algorithm.

ObjectivesTogether with the lysosomal storage diseases, NGLY1 deficiency is a congenital disorder of deglycosylation (NGLY1‐CDDG). Since the first report in 2012, 26 patients have been described. All but one were diagnosed by exome or genome sequencing; the remaining one was identified by finding an increased concentration of an urinary marker.The aim of this study was to describe the clinical, biochemical, and molecular features of the first Polish patient diagnosed with NGLY1‐CDDG, to provide an overview of the literature and to propose a diagnostic algorithm.ResultsA Polish patient presented with global developmental delay, hyperkinetic movement disorder, stagnation of head growth, hypolacrimia, elevated serum transaminases, and hypolipidemia in infancy. Whole exome sequencing revealed two heterozygous nonsense variants in the NGLY1 gene (a novel and an unreported). Literature review revealed global developmental disability in all reported patients, and hyperkinetic movements as well as alacrima/hypolacrima in nearly all.ConclusionsNGLY1‐CDDG should be considered in patients with developmental disability associated with a hyperkinetic movement disorder and alacrimia/hypolacrima. Absence of the latter two symptoms does not rule out this diagnosis.

An induced pluripotent stem cell line (TRNDi010-C) from a patient carrying a homozygous p.R401X mutation in the NGLY1 gene

NGLY1 deficiency is a rare inherited disorder caused by mutations in the NGLY1 gene encoding N-glycanase 1 that is a hydrolase for N-linked glycosylated proteins. An induced pluripotent stem cell (iPSC) line was generated from the dermal fibroblasts of a 16-year-old patient with homozygous mutation of p.R401X (c.1201 A>T) in the NGLY1 gene. Our iPSC model offers a useful resource to study the disease pathophysiology and to develop therapeutics for treatment of NGLY1 patients.

Generation of an induced pluripotent stem cell line (TRNDi002-B) from a patient carrying compound heterozygous p.Q208X and p.G310G mutations in the NGLY1 gene

NGLY1 deficiency is a rare genetic disease caused by mutations in the NGLY1 gene that encodes N-glycanase 1. The disease phenotype in patient cells is unclear. A human induced pluripotent stem cell (iPSC) line was generated from skin dermal fibroblasts of a patient with NGLY1 deficiency that has compound heterozygous mutations of a p.Q208X variant (c.622C > T) in exon 4 and a p.G310G variant (c.930C > T) in exon 6 of the NGLY1 gene. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for drug development to treat NGLY1 deficiency.

N-Glycanase 1 Deficiency Triggers Innate Immune Activation Through Dysregulated Mitophagy

Abstract The cytosolic N-glycanase 1 (Ngly1) is an evolutionarily conserved de-glycosylating enzyme, which plays a critical role in the quality control of newly synthesized glycoproteins. Misfolded N-glycoproteins retro-translocated from endoplasmic reticulum lumen to cytosol are deglycosylated by Ngly1 prior to proteasome degradation. Mutations in the human NGLY1 gene are associated with a severe rare congenital disorder characterized by global development delay, neurological abnormalities and liver diseases. The pathogenesis of the NGLY1-associated disease remains largely unknown. In this study, we observed an aberrant innate immune activation in both human and mouse Ngly1−/− cells, as evidenced by elevated expression of interferon-stimulated genes that are collectively known as the ‘type I interferon gene signature’. Using pharmacological inhibitors and RNAi knockdown, we identified the cytosolic DNA sensing pathway as the key innate immune pathway activated in Ngly1−/− cells. We also found that Ngly1−/− cells exhibited fragmented mitochondria and mtDNA leakage into the cytosol, which likely serve as the source for self DNA sensed by the innate immunity. Mitochondria are dynamic organelles, and aged or damaged mitochondria are cleared through mitophagy. We found that mitophagy was impaired in the absence of NGLY1. Together, our data reveal the first evidence of immune dysregulation associated with NGLY1 deficiency, as well as defects in mitophagy that likely lead to accumulation of damaged mitochondria, mtDNA leakage into the cytosol and activation of cytosolic DNA sensing pathway.

Lethality of mice bearing a knockout of the Ngly1-gene is partially rescued by the additional deletion of the Engase gene.

The cytoplasmic peptide:N-glycanase (Ngly1 in mammals) is a de-N-glycosylating enzyme that is highly conserved among eukaryotes. It was recently reported that subjects harboring mutations in the NGLY1 gene exhibited severe systemic symptoms (NGLY1-deficiency). While the enzyme obviously has a critical role in mammals, its precise function remains unclear. In this study, we analyzed Ngly1-deficient mice and found that they are embryonic lethal in C57BL/6 background. Surprisingly, the additional deletion of the gene encoding endo-β-N-acetylglucosaminidase (Engase), which is another de-N-glycosylating enzyme but leaves a single GlcNAc at glycosylated Asn residues, resulted in the partial rescue of the lethality of the Ngly1-deficient mice. Additionally, we also found that a change in the genetic background of C57BL/6 mice, produced by crossing the mice with an outbred mouse strain (ICR) could partially rescue the embryonic lethality of Ngly1-deficient mice. Viable Ngly1-deficient mice in a C57BL/6 and ICR mixed background, however, showed a very severe phenotype reminiscent of the symptoms of NGLY1-deficiency subjects. Again, many of those defects were strongly suppressed by the additional deletion of Engase in the C57BL/6 and ICR mixed background. The defects observed in Ngly1/Engase-deficient mice (C57BL/6 background) and Ngly1-deficient mice (C57BL/6 and ICR mixed background) closely resembled some of the symptoms of patients with an NGLY1-deficiency. These observations strongly suggest that the Ngly1- or Ngly1/Engase-deficient mice could serve as a valuable animal model for studies related to the pathogenesis of the NGLY1-deficiency, and that cytoplasmic ENGase represents one of the potential therapeutic targets for this genetic disorder.

Endo-β-N-acetylglucosaminidase forms N-GlcNAc protein aggregates during ER-associated degradation in Ngly1-defective cells

The cytoplasmic peptide:N-glycanase (PNGase; Ngly1 in mice) is a deglycosylating enzyme involved in the endoplasmic reticulum (ER)-associated degradation (ERAD) process. The precise role of Ngly1 in the ERAD process, however, remains unclear in mammals. The findings reported herein, using mouse embryonic fibroblast (MEF) cells, that the ablation of Ngly1 causes dysregulation of the ERAD process. Interestingly, not only delayed degradation but also the deglycosylation of a misfolded glycoprotein was observed in Ngly1(-/-) MEF cells. The unconventional deglycosylation reaction was found to be catalyzed by the cytosolic endo-β-N-acetylglucosaminidase (ENGase), generating aggregation-prone N-GlcNAc proteins. The ERAD dysregulation in cells lacking Ngly1 was restored by the additional knockout of ENGase gene. Thus, our study underscores the functional importance of Ngly1 in the ERAD process and provides a potential mechanism underlying the phenotypic consequences of a newly emerging genetic disorder caused by mutation of the human NGLY1 gene.