Natural history of NGLY1 deficiency: motor function & clinical features

N-glycanase 1 (NGLY1) deficiency is a debilitating, ultra-rare autosomal recessive disorder caused by loss of function of NGLY1, a cytosolic enzyme that deglycosylates other proteins. It is characterized by severe global developmental delay and/or intellectual disability, hyperkinetic movement disorder, transient elevation of transaminases, (hypo)alacrima and progressive, diffuse, length-dependent sensorimotor polyneuropathy. A prospective natural history study (NHS) was conducted to elucidate clinical features and disease course. Twenty-nine participants were enrolled (15 onsite, 14 remotely) and followed for up to 32months, representing ~29% of the ~100 patients identified worldwide. Participants exhibited profound developmental delays, with almost all developmental quotients below 20 on the Mullen Scales of Early Learning, well below the normative score of 100. Increased difficulties with sitting and standing suggested decline in motor function over time. Most patients presented with (hypo)alacrima and reduced sweat response. Pediatric quality of life was poor except for emotional function. Language/communication and motor skill problems including hand use were reported by caregivers as the most bothersome symptoms. Levels of the substrate biomarker, GlcNAc-Asn (aspartylglucosamine; GNA), were consistently elevated in all participants over time, independent of age. Liver enzymes were elevated for some participants but improved especially in younger patients and did not reach levels indicating severe liver disease. Three participants died during the study period. Data from this NHS informs selection of endpoints and assessments for future clinical trials for NGLY1 deficiency interventions. Potential endpoints include GNA biomarker levels, neurocognitive assessments, autonomic and motor function (particularly hand use), (hypo)alacrima and quality of life.

AAV9-NGLY1 gene replacement therapy improves phenotypic and biomarker endpoints in a rat model of NGLY1 Deficiency

Substrate-derived biomarkers are necessary in slowly progressing monogenetic diseases caused by single-enzyme deficiencies to identify affected patients and serve as surrogate markers for therapy response. N-glycanase 1 (NGLY1) deficiency is an ultra-rare autosomal recessive disorder characterized by developmental delay, peripheral neuropathy, elevated liver transaminases, hyperkinetic movement disorder and (hypo)-alacrima. We demonstrate that N-acetylglucosamine-asparagine (GlcNAc-Asn; GNA), is the analyte most closely associated with NGLY1 deficiency, showing consistent separation in levels between patients and controls. GNA accumulation is directly linked to the absence of functional NGLY1, presenting strong potential for its use as a biomarker. In agreement, a quantitative liquid chromatography with tandem mass spectrometry assay, developed to assess GNA from 3 to 3000 ng/ml, showed that it is conserved as a marker for loss of NGLY1 function in NGLY1-deficient cell lines, rodents (urine, cerebrospinal fluid, plasma and tissues) and patients (plasma and urine). Elevated GNA levels differentiate patients from controls, are stable over time and correlate with changes in NGLY1 activity. GNA as a biomarker has the potential to identify and validate patients with NGLY1 deficiency, act as a direct pharmacodynamic marker and serve as a potential surrogate endpoint in clinical trials.

N-glycanase 1 (NGLY1) Deficiency is an ultra-rare autosomal recessive disorder of deglycosylation caused by loss-of-function mutations in the NGLY1 gene. Patients present with developmental delay, intellectual disability, hyperkinetic movement disorder, elevated liver enzymes, (hypo)alacrima, and peripheral neuropathy. Despite supportive care, many experience early neurological deterioration, with loss of previously attained motor skills by adolescence. Additionally, life-threatening complications are not uncommon, and the published median lifespan of patients is ~13 years. The pathophysiology of NGLY1 Deficiency remains poorly understood, in part due to limited long-term studies in animal models. Notably, Ngly1⁻/⁻ mice (C57BL/6) are embryonically lethal, and prior characterization of Ngly1⁻/⁻ rats was restricted to young adult rat (~7 months old), leaving late-onset phenotypes and potential lifespan reduction unexplored. In the study reported here, longitudinal assessments of phenotypes in Ngly1⁻/⁻ rats were conducted alongside Ngly1⁺/⁻ and Ngly1⁺/⁺ control rats. Survival, motor function, biochemical biomarkers, and brain histopathology were examined in the rats from approximately 6 months to 17–18 months of age. Ngly1⁻/⁻ rats exhibited markedly reduced lifespan, progressive neurological decline, and decreased quality of life compared with Ngly1⁺/⁻ and Ngly1⁺/⁺ rats. By 9–10 months of age, ~50% of the Ngly1⁻/⁻ rats had either died or met humane euthanasia criteria due to a severe decline in health. Surviving animals displayed phenotypes mirroring human NGLY1 Deficiency disease progression, such as worsening motor deficits (~92% reduction in rotarod latency and ~82% reduction in rearing) and wide-spread neuroinflammation in multiple brain regions. In contrast, Ngly1⁺/⁻ and Ngly1⁺/⁺ littermates remained healthy and exhibited normal lifespan and aging profiles. Furthermore, histopathological examination of Ngly1⁻/⁻ rats identified significant neuropathological abnormalities that were not present in the control cohorts, including loss of peripheral axons and spinal motor neurons. The findings reported here demonstrate that Ngly1⁻/⁻ rats recapitulate the severe, progressive course of NGLY1 Deficiency, including neurodegenerative deterioration, motor deficits, and premature mortality. This extended longitudinal assessment of Ngly1⁻/⁻ rats provides important insights into disease progression and the shortened lifespan reported for human patients.

Patients with mutations in NGLY1 cannot make tears (alacrima), have global developmental delay, movement disorder and liver dysfunction. N‐glycanase 1 (NGLY1) de‐glycosylates misfolded N‐glycosylated proteins in the cytoplasm as part of the ERAD pathway prior to their proteasomal degradation. Surprisingly, NGLY1‐deficient patient cells do not accumulate cytoplasmic misfolded N‐glycoproteins, suggesting a more complex function. Interestingly, new studies show NGLY1 is needed for transcription factor NFE2L1/NRF1 activation and normal mitochondrial function.Using WT and NGLY1‐deficient mouse embryonic fibroblasts (MEFs) we found that NGLY1‐deficient cells were resistant to hypotonic lysis compared to WT. The same was seen in NGLY1‐patient fibroblasts. Additionally, MEFs deficient in both NGLY1 and ENGase (another deglycosylation enzyme) were even more resistant to hypotonic lysis compared to NGLY1‐deficient cells. Water influx and cell swelling precedes cell lysis. We found that NGLY1‐deficient MEFs swell slower than WT MEFs. Since aquaporins (AQP) transport water, we hypothesized that AQP levels might be disrupted in NGLY1‐deficient cells. We found both AQP1 mRNA and protein were reduced in NGLY1‐deficient MEFs. shRNA knockdown of AQP1 in WT MEFs decreased hypotonic lysis, suggesting AQP1 is associated with hypotonic lysis. NGLY1 shRNA and CRISPR studies confirmed that NGLY1 regulates AQP1 levels and hypotonic cell lysis. Preliminary studies show that complementing NGLY1‐deficient MEFs with NGLY1 increases AQP1 and restores hypotonic lysis. Current efforts are directed towards understanding whether NGLY1 enzyme activity is necessary for AQP regulation. 13 types of AQP are expressed in different cell types. Our preliminary data suggest that AQP11 levels are disrupted in NGLY1‐deficient patient cells and CRISPR cells, suggesting that NGLY1 might regulate different types of AQP.We have identified a novel function of NGLY1, i.e. to directly/indirectly regulate AQPs. This finding may relate to NGLY1‐deficient patients' inability to make tears. Future efforts will try to identify NGLY1‐dependent transcription factors responsible for regulating AQPs. This work is supported by the Bertrand Might Research Fund and NGLY1.org.Support or Funding InformationThis work is supported by the Bertrand Might Research Fund.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

N-Glycanase 1 Regulates Aquaporins Independent of Its Enzymatic Activity

GS-100 is an AAV9 gene replacement therapy for the treatment of N-glycanase 1 (NGLY1) deficiency, an ultra-rare, recessive disorder characterized by developmental delay, intellectual disability, hyperkinetic movement disorder, elevated liver enzymes, (hypo)alacrima, and peripheral neuropathy. In pharmacology studies using Ngly1 -/- rats, intracerebroventricular (i.c.v.) administration of GS-100 showed dose-dependent biodistribution, increased hNGLY1 mRNA expression, reduced disease biomarker N-acetylglucosamine-asparagine (GNA), improved motor function, and prevented phenotype progression. Reductions in GNA within central nervous system (CNS) tissues and cerebrospinal fluid (CSF) correlated significantly with improvements, suggesting that CSF GNA may serve as a clinical biomarker of therapeutic activity. Safety studies conducted in wild-type rats and non-human primates (NHPs) indicated no adverse clinical signs, mortality, body weight changes, or negative neurobehavioral/nerve conduction effects at or near the clinical starting dose of 4e14 vector genome (vg)/individual. In addition, GS-100 biodistribution was widespread in CNS and peripheral tissues. These nonclinical data supported the Food and Drug Administration approval of an Investigational New Drug application for a first-in-human phase 1/2/3 dose-escalation clinical trial. The trial's co-primary endpoints include changes in CSF GNA biomarker levels and improvements or stabilization of motor function to assess the safety and efficacy of GS-100 administered i.c.v. in patients with NGLY1 deficiency.

The purpose of the research presented in this chapter is to investigate if virtual reality (VR) neurocognitive, executive, and motor function assessment tools are susceptible to practice and fatigue effects similar to those currently used in a clinical practice. Fifteen athletically active and neurologically normal Penn State students participated in a VR “practice effect” study. Another 15 Penn State football players participated in an “effect of fatigue” study on neurocognitive, balance, and executive functions. Subjects performed VR tests on several occasions. The statistical analysis was conducted to examine the VR measures as a function of testing session (practice effect) and physical fatigue (prior to and after full contact practices). The number and type of the full contacts during the practices were assessed via a specially developed observational chart. There are several major findings of interest. First, all subjects reported the “sense of presence” and “significant mental effort” while performing the VR tests. Second, neither effect of testing day (p > 0.05) nor effect of VR testing modality (p > 0.05) was revealed by ANOVA. Third, physical fatigue did not influence the VR measures in the majority of football players under study (p > 0.05). However, there was a reduction in several VR performance measures in football players who sustained prior concussive injuries. The findings show that VR testing modalities implemented in this study and aimed to evaluate neurocognitive (spatial memory, attention), motor (balance), and executive functions may be used as a complementary tool in a clinical practice. VR testing modalities under laboratory conditions are easily transferable into field conditions, and can potentially be used as the side-line evaluation of subjects at risk for concussion.

N-glycanase 1 (NGLY1) Deficiency is a rare monogenic multi-system disorder first described in 2014. NGLY1 is evolutionarily conserved in model organisms. Here we conducted a natural history study and chemical-modifier screen on the Drosophila melanogaster NGLY1 homolog, Pngl. We generated a new fly model of NGLY1 Deficiency, engineered with a nonsense mutation in Pngl at codon 420 that results in a truncation of the C-terminal carbohydrate-binding PAW domain. Homozygous mutant animals exhibit global development delay, pupal lethality and small body size as adults. We developed a 96-well-plate, image-based, quantitative assay of Drosophila larval size for use in a screen of the 2,650-member Microsource Spectrum compound library of FDA approved drugs, bioactive tool compounds, and natural products. We found that the cholesterol-derived ecdysteroid molting hormone 20-hydroxyecdysone (20E) partially rescued the global developmental delay in mutant homozygotes. Targeted expression of a human NGLY1 transgene to tissues involved in ecdysteroidogenesis, e.g., prothoracic gland, also partially rescues global developmental delay in mutant homozygotes. Finally, the proteasome inhibitor bortezomib is a potent enhancer of global developmental delay in our fly model, evidence of a defective proteasome “bounce-back” response that is also observed in nematode and cellular models of NGLY1 Deficiency. Together, these results demonstrate the therapeutic relevance of a new fly model of NGLY1 Deficiency for drug discovery and gene modifier screens.

Background Our previous studies confirmed that human Wharton's Jelly stem cell (hWJSC) transplantation improved motor function in children with spastic cerebral palsy (CP). This study investigated the dose-effect relationship between the transplanted cell dosage and efficacy in CP children. Methods CP children who received one- or two-course (four or eight times lumbar puncture, 4 or 8 × 107 hWJSCs) cell therapy were recruited into this study. Assessments of motor function were performed according to scales for gross motor function measurement (GMFM) and fine motor function measurement (FMFM). The measurement data obtained in the two different groups were analyzed by t-test. Univariate repeated measures analysis of variance was used to compare the data obtained at baseline and 6 or 12 months posttransplantation and met the conditions for Mauchly's sphericity test. Results The results for fifty-seven pediatric CP patients (including 35 male and 22 female patients) who completed follow-up showed that gross and fine motor functions improved after cell therapy. Interestingly, the GMFM and FMFM scores in patients who received one course of transplantation were significantly increased at 6 months after treatment. Moreover, another course of transplantation further improved gross and fine motor function in children. The scores for GMFM and FMFM were significantly higher at 6 months posttransplantation than at baseline and showed a linear upward trend. There was no gender difference in GMFM. Interestingly, there was a significant difference between male and female patients in the B and C dimensions of FMFM. These results reveal a gender-related susceptibility to stem cell therapy, especially for movement capability of the upper extremity joint and grasping ability. Similarly, in the group aged ≤3 years old, the improvement observed in dimension A (lying and rolling) of GMFM was nearly exponential and showed a quadratic trend. The results for FMFM were similar to those for GMFM. Moreover, the improvement in motor function was not age dependent. Conclusions In this study, our data collectively reveal that CP children display sex- or age-dependent responses to hWJSC therapy; these results shed light on the clinical utility of this approach in specific populations.

Neurocognitive dysfunction and brain injury on magnetic resonance imaging (MRI) are common in patients with systemic lupus erythematosus (SLE) and are associated with increased morbidity and mortality. However, brain MRI is expensive, is restricted by payers, and requires high expertise. Neurocognitive assessment is an easily available, safe, and inexpensive clinical tool that may select patients needing brain MRI. In this cross-sectional and controlled study, 76 SLE patients (69 women, age 37 ± 12 years) and 26 age and gender-matched healthy subjects (22 women, age 34 ± 11 years) underwent assessment of attention, memory, processing speed, executive function, motor function, and global neurocognitive function. All subjects underwent brain MRI with T1-weighted, fluid-attenuated inversion recovery (FLAIR), and diffusion-weighted imaging. Hemispheric and whole brain lesion load in cm3 were determined using semi-automated methods. Neurocognitive z-scores in all clinical domains were significantly lower and whole brain and right and left hemispheres brain lesion load were significantly greater in patients than in controls (all p ≤ 0.02). There was significant correlation between neurocognitive z-scores in all domains and whole brain lesion load: processing speed (r = - 0.46; p < 0.0001), attention (r = - 0.42; p < 0.001), memory (r = - 0.40; p = 0.0004), executive function (r = - 0.25; p = 0.03), motor function (r = - 0.25; p = 0.05), and global neurocognitive function (r = - 0.38; p = 0.006). Similar correlations were found for brain hemisphere lesion loads (all p ≤ 0.05). These correlations were strengthened when adjusted for glucocorticoid therapy and SLE disease activity index. Finally, global neurocognitive z-score and erythrosedimentation rate were the only independent predictors of whole brain lesion load (both p ≤ 0.007). Neurocognitive measures and brain lesion load are worse in SLE patients than in controls. In SLE patients, neurocognitive z-scores correlate negatively with and independently predict brain lesion load. Therefore, neurocognitive testing may be an effective clinical tool to select patients needing brain MRI.

Background: Neurocognitive dysfunction and brain injury on MRI are common in patients with systemic lupus erythematosus (SLE) and are associated with increased morbidity and mortality. However, brain MRI is expensive, non-specific, and does not necessarily predict neurocognitive dysfunction. In contrast, neurocognitive assessment is a clinical tool that is easily available, inexpensive, safe, and may better select patients who require an MRI or other cerebrovascular imaging. Methods: 76 patients with SLE [71 women, age 36±12 years] and 26 age-and-gender matched healthy subjects [22 women, age 34±11 years] underwent neurocognitive testing for assessment of global neurocognitive function, attention, memory, processing speed, executive function, and motor function (expressed as z-scores). All 102 subjects underwent brain MRI. T1 weighted, fluid attenuated inversion recovery, and diffusion weighted images were obtained. Whole brain and hemispheric lesion load in cm 3 were determined using a semi-automated method. Results: All assessments of neurocognitive function were significantly worse in SLE patients than in controls, including global cognitive function (-1.82±1.90 versus -0.07±0.52,), attention (-1.80±2. versus -0.06±0.82,), memory (-1.23±1.15 versus -0.056±0.81 ), processing speed (-1.38 ±1.63 versus -0.06±0.93 ), executive function (-2.39±2.96 versus -0.10±0.79,) and motor function (-3.14±5.90 versus -0.06±0.63) (all p≤0.003). Also, white matter brain lesion load of the whole brain, left hemisphere, and right hemisphere were greater in SLE patients than in controls (1.10±2.65 versus 0.10±0.28 cm 3 , 0.41±0.98 versus 0.04±0.13 cm 3 and 0.68±1.97 versus 0.057±0.16 cm 3 , respectively, all p≤0.02]. Neurocognitive scores in each domain were significantly negatively correlated with brain lesion load of the whole brain, left and right hemispheres (r = 0.33 - 0.46, all p≤0.02, Pearson Correlation Coefficients ) . Conclusions: In SLE patients each neurocognitive domain score correlates negatively with brain lesion on MRI. These findings suggest that neurocognitive testing may be an effective initial clinical tool to assess for ischemic brain injury in SLE patients.

Motor impairments are frequent both at and before diagnosis. In childhood, impairments in general fine and gross motor function are among others identified using test batteries, and while elements of coordination are assessed in onset schizophrenia, the assessment of general motor functions is absent. Thus, we aimed to assess general motor function including childhood motor function in adolescents with schizophrenia in comparison with healthy controls and examine clinical correlates to general motor function. General fine and gross motor function was assessed using two standardized age-normed test batteries and a questionnaire in 25 adolescents with schizophrenia compared with age and gender-matched controls using t-test and χ2 -test. Stepwise linear regression assessed potential developmental predictors on motor function including complications during childbirth, reported childhood motor function, executive function including false discovery rate q-values. Associations with schizophrenia symptom severity, executive function, cognitive function were assessed using Pearson's correlation and the impact of antipsychotic medication using t-test. All measures of motor function but one significantly differentiated adolescents with schizophrenia from healthy controls. The presence of schizophrenia (β =4.41, β=10.96), explained the main part of the variance however, childhood motor function (β=.08) also added significantly to motor function. Executive function (β=-.45) was important for childhood motor function. Severity of schizophrenia was associated with strength (p < .0011) and manual coordination (p=.0295), and receiving antipsychotics affected manual dexterity (p=.0378). The documentation of significant differences in general motor function in early onset schizophrenia compared with healthy controls highlights the need for general motor assessments and potential interventions.

Among older adults, both olfaction and motor function predict future cognitive decline and dementia, suggesting potential shared causal pathways. However, it is not known whether olfactory and motor function are independently related in late life. We assessed cross-sectional associations of olfaction with motor and cognitive function, using concurrent data on olfactory function, mobility, balance, fine motor function, manual dexterity, and cognition in 163 Baltimore Longitudinal Study of Aging participants aged 60 and older without common neurological diseases (n = 114 with available cognitive data). Using multiple linear regression, we adjusted for age, sex, race, smoking history, height, and weight for mobility and balance, and education for cognition. We used multiple linear regression to test whether olfaction-motor associations were independent of cognition and depressive symptoms. Olfactory scores were significantly associated with mobility (usual gait speed, rapid gait speed, 400-m walk time, and Health ABC Physical Performance Battery score), balance, fine motor function, and manual dexterity (all p < .05). In those with available cognitive data, additional adjustment for depressive symptoms, verbal memory, or visuoperceptual speed demonstrated especially strong independent relationships with challenging motor tasks such as 400-m walk and nondominant hand manual dexterity (p < .005). This study demonstrates for the first time that, in older adults, olfactory function is associated with mobility, balance, fine motor function, and manual dexterity, and independent of cognitive function, with challenging upper and lower extremity motor function tasks. Longitudinal studies are needed to determine if olfactory performance predicts future mobility and functional decline.

To examine interrater agreement and validity of the Functional Communication Classification System (FCCS) for young children with cerebral palsy (CP) aged 2 or 3 years. Speech-language pathologist (SLP) and parent FCCS ratings for 31 children with CP (aged 2y, n=16; aged 3y, n=15; 18 males, 13 females) were examined for interrater agreement using a weighted Cohen's kappa statistic. Relationships between FCCS (SLP) ratings and: (1) concurrent validity with the Language Use Inventory, a standardized pragmatic assessment for children aged 18 to 47 months, (2) gross motor and fine motor function, (3) associated impairments (visual and intellectual), and (4) primary expressive communication mode were examined using Spearman's correlation coefficients. Almost perfect interrater agreement between SLP and parent FCCS ratings were found (kw =0.94). Correlations with FCCS (SLP) were excellent for pragmatic function (rs =-0.83,p<0.001), intellectual function (rs =0.89, p<0.001), and primary expressive communication mode (rs =0.92,p<0.001). Correlations were good for gross motor function (rs =0.72,p<0.001) and visual impairment (rs =0.70,p<0.001) and fair for fine motor function (rs =0.53,p<0.002). Analysis was unwarranted for epilepsy (n=1 out of 31) and hearing-associated impairments (n=0 out of 31). The FCCS has excellent interrater agreement and validity for communication classification of children with CP aged 2 or 3 years and is highly suitable for surveillance and research purposes. What this paper adds The Functional Communication Classification System (FCCS) is a valid instrument for children with cerebral palsy (CP) aged 2 or 3 years. Excellent agreement exists between speech-language pathologist and parent FCCS ratings. The FCCS has excellent correlation with intelligence, pragmatic function, and primary expressive mode. Stronger correlations with the Gross Motor Function Classification System and vision exist for children aged 2 or 3 years. Weaker correlations with manual ability exist when compared to older children with CP.