Progressive Neurodegeneration, Motor Decline, and Premature Mortality in Aging Ngly1 Deficient Rats

Progressive Neurodegeneration, Motor Decline, and Premature Mortality in Aging Ngly1 Deficient Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Progressive Neurodegeneration, Motor Decline, and Premature Mortality in Aging Ngly1 Deficient Rats Lei Zhu, Selina Dwight, William Mueller, Becky Schweighardt This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7014298/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Feb, 2026 Read the published version in Orphanet Journal of Rare Diseases → Version 1 posted 6 You are reading this latest preprint version Abstract N-glycanase 1 (NGLY1) Deficiency is an ultra-rare autosomal recessive disorder of deglycosylation caused by loss-of-function mutations in the NGLY1 gene. Patient symptoms are characterized by developmental delay, intellectual disability, hyperkinetic movement disorder, elevated liver enzymes, (hypo)alacrima, and peripheral neuropathy. Despite supportive care, affected individuals often exhibit neurological deterioration at a young age, with caregivers reporting loss of previously attained motor skills by adolescence. Additionally, life-threatening complications are not uncommon, and the published median lifespan of patients is ~ 15 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) before sacrifice, leaving any late-onset disease phenotypes or understanding of the potential for shortened lifespan unexamined. In the study reported here, longitudinal assessments of phenotypes in Ngly1 −/− rats were conducted alongside Ngly1 +/− and Ngly1 +/+ controls. Survival, motor function, biochemical disease biomarkers, and histopathology of brain tissues were monitored in the rats from approximately 6 months to 17–18 months of age. Results : Ngly1 −/− rats exhibited markedly reduced lifespan and progressive decline in both neurological behavior and 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 Ngly1 −/− rats showed other phenotypes mirroring human NGLY1 Deficiency disease progression, such as worsening motor deficits and wide-spread neuroinflammation. In contrast, heterozygous and wild-type littermates remained healthy and exhibited normal lifespan and aging profiles. Furthermore, histopathological examination of Ngly1 −/− rats identified significant neuropathological abnormalities not present in the control cohorts, including loss of peripheral axons and spinal motor neurons. Conclusion : 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 assessment of phenotypes and histology in Ngly1 −/− rats over an extended period of time provides valuable insights with respect to disease progression and lifespan in human patients. NGLY1 NGLY1 Deficiency Rare diseases Motor function CNS Mortality Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction NGLY1 Deficiency (OMIM #615273) is an ultra-rare autosomal recessive genetic disease caused by biallelic mutations in the NGLY1 gene. NGLY1 encodes N-glycanase 1, a conserved cytosolic enzyme that removes N-linked glycans from misfolded glycoproteins during endoplasmic reticulum-associated degradation (ERAD) [ 1 ]. Loss of NGLY1 disrupts this critical protein quality-control pathway, leading to accumulation of undegraded glycoproteins and widespread cellular stress [ 2 , 3 , 4 ]. Clinically, NGLY1 Deficiency presents as a suite of complex neurodevelopmental phenotypes with global developmental delay and/or intellectual disability, a hyperkinetic movement disorder, transient elevation of liver transaminases, (hypo)alacrima (insufficient or absent tear production), and a chronic diffuse sensorimotor neuropathy that affects the nerves in a length-dependent manner. As children grow, a characteristic hyperkinetic movement disorder (chorea/dystonia) emerges frequently alongside epilepsy [ 6 ]. Peripheral neuropathy develops progressively, often manifesting as areflexia and distal weakness, and other musculoskeletal complications such as scoliosis and contractures can arise [ 7 , 8 , 9 ]. Notably, while early mortality was not prominent in initial case studies [ 10 , 11 ], recent longitudinal data suggest a limited lifespan in NGLY1 Deficiency patients, with a mean age of death of 13 (reported in 2022) [ 9 ], recently increasing to 14.6 years [unpublished data, Grace Science Foundation]. Together, these observations suggest that NGLY1 Deficiency results in progressive deterioration and early mortality in patients. Animal models are crucial for understanding the disease mechanism and testing therapies, yet modeling NGLY1 Deficiency has been challenging. Ngly1 -knockout mice in a C57BL/6 background exhibit embryonic lethality [ 12 ] and thus preclude postnatal studies. Given the limitations in mice, an Ngly1 −/− rat model was developed to enable postnatal and longitudinal investigations [ 13 ]. This hurdle was later partially overcome by using a mixed (JF1/B6) genetic background [ 14 ]. This rat model showed high early mortality (70%) within the first three weeks after birth, stabilizing after weaning (21 days), and recapitulated key clinical features of NGLY1 Deficiency, including failure to thrive, peripheral neuropathy, hyperkinetic movements, and delayed cognitive development [ 13 ] [ 14 ]. Histopathological analyses in young adult Ngly1 −/− rats (up to ~ 7 months old) revealed neurodegenerative changes such as neuron loss in the thalamus, astrogliosis, microgliosis, and peripheral nerve axonopathy [ 15 ]. While providing valuable insights into disease pathology, these early studies were not designed to follow adult animals. Consequently, little is known about the trajectory of NGLY1 Deficiency into late adulthood in an animal model, including whether new phenotypes emerge or if the pathology worsens with age. This gap in understanding is relevant given that some NGLY1 Deficiency patients live beyond their teenage years despite the shortened mean lifespan associated with the disease [ 9 ]. An aging study was therefore conducted in the Ngly1 −/− rat model to inform late-stage disease mechanisms with the eventual aim of developing and evaluating interventions that slow disease progression and extend lifespan. A longitudinal study of Ngly1 −/− rats was conducted to help characterize the course of NGLY1 Deficiency in aging animals. In this study, a cohort of homozygous Ngly1 −/− rats was followed from ~ 6 months through 17–18 months of age alongside Ngly1 +/− (heterozygous) and Ngly1 +/+ (wild-type) littermate control cohorts. Throughout this period, animals underwent periodic evaluations of motor behavior (rotarod, open field locomotor activity and rearing, and functional observational battery), assessments of the NGLY1 Deficiency biomarker, GlcNAc-Asn (GNA in both plasma and CSF levels [ 16 ]), and clinical indicators of health (body weight, survival, cage-side/clinical observation, hematology, and clinical chemistry). At end of life, comprehensive histopathological examinations were performed on each cohort, focusing on the nervous system (brain, spinal cord, peripheral nerves) and other organs relevant to NGLY1 Deficiency disease pathology. Here, we report that NGLY1 Deficiency in a rat model of the disease leads to severe premature aging phenotypes, including significant motor decline and late-onset neuromuscular deterioration, as well as mid-life mortality, thereby mirroring the human disease in both severity and progression. Our findings extend the phenotypic characterization of Ngly1 −/− animals beyond early adulthood and underscore the utility of this rat model for studying the pathogenesis and treatment of NGLY1 Deficiency in its advanced stages. Results Ngly1 ⁻/⁻ Rats Exhibit Progressive Neurological Phenotypes, Reduced Body Weight, And Premature Mortality as They Age At study initiation (~ 6 months of age), animals were assigned to one of three cohorts: wild-type ( Ngly1 +/+ ; N = 10, 5 males/5 females), heterozygous ( Ngly1 +/− ; N = 10, 5 males/5 females), or homozygous ( Ngly1 −/− ; N = 18, 9 males/9 females). Additional animals were included in the Ngly1 ⁻/⁻ group to account for anticipated mortality-related loss of animal number. Male Ngly1 ⁻/⁻ rats weighed significantly less than Ngly1 ⁺/⁺ and Ngly1 ⁺/⁻ males throughout the study (p < 0.05, linear mixed model), whereas female Ngly1 ⁻/⁻ body weights did not differ from controls (Fig. 1 ). Survival was also assessed for each rat in the 3 cohorts. Based on prior observations in Ngly1 ⁻/⁻ rats, litter sizes were not counted during the first few postnatal days to avoid disturbing the nests and potentially increasing postnatal mortality. Due to this, overall survival in the postnatal period from time of birth could not be assessed. After genotyping at postnatal day (PND) 15 and weaning at PND 21, the survival was 100% in all genotypes until ~ 8.5 months of age, at which point Ngly1 ⁻/⁻ rats exhibited a premature mortality that was not observed in either Ngly1 ⁺/⁺ or Ngly1 ⁺/⁻ cohorts. Eight of 18 Ngly1 ⁻/⁻ rats (44%) died or required humane euthanasia due to severe decline in cage side/clinical observations between PND 255 and 299 (~ 8.5–10 months of age). The remaining 10 Ngly1 ⁻/⁻ animals showed various signs of severe health deterioration and were humanely euthanized at 12 months and their tissues preserved. Ngly1 ⁻/⁻ Rats Exhibit Severe and Persistent Motor Deficits in the Rotarod Assessment, s The rotarod test measures the latency for each rat to fall from a rotating rod and is a measure of motor function [ 17 , 18 ]. Rotarod performance was assessed at 6.9 and 10.4 months of age for Ngly1 +/+ , Ngly1 +/− , and Ngly1 −/− rats and was determined to be significantly impaired in Ngly1 −/− rats compared to both Ngly1 +/+ and Ngly1 +/− rats. At ~ 6.9 months of age, Ngly1 −/− rats exhibited a latency to fall of 8.9 ± 3.7 seconds, which was significantly shorter than Ngly1 +/+ (81.4 ± 11.0 s, p = 3.4×10⁻⁴) and Ngly1 +/− (103.2 ± 18.6 s, p = 4.0×10⁻⁵) rats. Similarly, at 10.4 months, Ngly1 −/− rats maintained significantly lower performance (6.4 ± 0.2 seconds) than Ngly1 +/+ and Ngly1 +/− rats which sustained motor coordination with latencies of 80.4 ± 10.9 and 81.1 ± 11.6 seconds, respectively ( p < 4×10⁻⁷). No significant differences in rotarod performance were observed between Ngly1 +/+ and Ngly1 +/− rats at any tested time point, including at 16.6 months of age, indicating that the severe motor deficit is specific to complete loss of NGLY1. Data from Ngly1 −/− rats were not available for a comparison of rotarod performance at 16.6 months due to the premature mortality of some animals and the significant deterioration of the surviving animals. Ngly1 ⁻/⁻ Rats Show Significantly Reduced Locomotor Activity and Rearing at Compared with Ngly1 +/+ and Ngly1 +/− Rats Locomotor activity was assessed for all 3 rat cohorts at 6.9 and 9 months of age in an open field test using a Hamilton-Kinder enclosure, where horizontal (basic movement) and vertical (rearing) movements were recorded via infrared beam breaks over a 15-minute session. In the open field test, basic movement and number of rearings were both significantly lower in Ngly1 −/− rats compared to Ngly1 +/+ and Ngly1 +/− rats at both 6.9-month and 9-month time points. No significant difference in basic movements or number of rearings was observed between Ngly1 +/+ and Ngly1 +/− rats at any of the tested time points (6.9, 9, and 10.8 months; Fig. 4 ). At 6.9 months of age, Ngly1 ⁻/⁻ rats showed markedly lower basic movements (17,909.7 ± 1,193.5) compared to Ngly1 ⁺/⁺ (27,195.0 ± 2,463.6, p = 0.0087) and Ngly1 ⁺/⁻ (26,579.2 ± 2,495.3, p = 0.0194) rats. A similar pattern was observed in rearing activity, where Ngly1 ⁻/⁻ rats displayed a significantly lower number of rearings (204.4 ± 25.2) than both Ngly1 ⁺/⁺ (748.6 ± 122.0, p = 0.0007) and Ngly1 ⁺/⁻ (813.9 ± 173.3, p = 0.0006) cohorts. At 9.0 months of age, Ngly1 ⁻/⁻ rats continued to exhibit significantly reduced basic movement (18,266.8 ± 1,431.5) compared to Ngly1 ⁺/⁺ (31,616.8 ± 2,285.0, p = 0.0003) and Ngly1 ⁺/⁻ (28,474.9 ± 2,547.9, p = 0.007) rats. As was also observed for rats 6.9 months of age, Ngly1 ⁻/⁻ rats at 9.0 months of age displayed a significantly lower number of rearings (176.8 ± 27.8) than both Ngly1 ⁺/⁺ (1,013.2 ± 138.0, p = 5 ×10⁻ 5 ) and Ngly1 ⁺/⁻ (879.1 ± 137.9, p = 0.0003) rats. Data from Ngly1 −/− rats were not available for a comparison of basic movement and number of rearings at 10.8 months due to the premature mortality of some animals and the significant deterioration of the surviving animals, but Ngly1 ⁺/⁺ and Ngly1 ⁺/⁻ rats were tested at this time point. No significant differences in either basic movements or number of rearings were observed between Ngly1 ⁺/⁺ and Ngly1 ⁺/⁻ rats at any tested age, suggesting that loss of a single NGLY1 allele does not impair gross locomotor behavior as assessed in the locomotor open field test. In the Functional Observational Battery (FOB), a test of neurobehavioral behavior, Ngly1 ⁻ / ⁻ rats exhibited impairments in rearing (as assessed for 1-minute by a scorer), righting reflexes, and gait/mobility when compared to Ngly1 ⁺/⁺ and Ngly1 ⁺/⁻ rats at approximately 7 and 9 months of age ( Supplemental Fig. 1 ). No significant differences were observed in other FOB parameters (Data not shown). As with the locomotor open field test, there were no differences detected between Ngly1 ⁺ / ⁺ and Ngly1 ⁺ / ⁻ rats at any time point. ( Supplemental figure x, Data not shown ) Based on cage side clinical observations, Ngly1 ⁻ / ⁻ rats displayed a consistent neurological phenotype at study initiation (~ 6 months of age) that was consistent with the previous characterization of this Ngly1 deficient rat model (Asahina et al 2020, Zhu et al 2022). Specifically, the Ngly1 −/− rats exhibited uncoordinated gait, hindlimb weakness (often asymmetric), bruxism, splayed limbs, head tilt, and labored breathing. Additional observations exclusive to Ngly1 ⁻ / ⁻ animals in this study included malocclusion, thin body condition, low head carriage, skin discoloration with scabbing, and dental discoloration. Overall, these phenotypes progressively worsened over time. Ngly1 ⁻/⁻ rats Exhibit Consistently Elevated GNA levels in both plasma and CSF. Longitudinal analyses of GNA levels in both cerebrospinal fluid (CSF) and plasma were conducted in Ngly1 −/− , Ngly1 +/+ , and Ngly1 +/− rats at the following time points: 6.2–7.1 months, 8.7–9.2 months, and 10.6–11.3 months (Fig. 4 ). Throughout the observation period, plasma GNA levels were consistently higher in Ngly1 −/− rats compared with either Ngly1 +/+ or Ngly1 +/− rats at each time point. Interestingly, GNA levels in the plasma of Ngly1 −/− rats demonstrated an age-dependent increase from 6.5 months of age to their death or humane euthanasia at 12 months of age, while plasma GNA levels in Ngly1 +/+ and Ngly1 +/− rats remained at similar, low levels throughout the study. Ngly1 −/− rats also exhibited consistently elevated CSF GNA levels (252.2 ± 6.3 ng/mL, 224.5 ± 6.6 ng/mL, 336 ± 73 ng/mL) compared to Ngly1 +/+ (27.5 ± 1.3 ng/mL, 23.6 ± 0.9 ng/mL, and 35.6 ± 2.9 ng/mL)and Ngly1 +/− (46.5 ± 1.5 ng/mL, 43.7 ± 1.8 ng/mL, 43.1 ± 2.7 ng/mL) rats across all 3 evaluated time points. In contrast to plasma GNA levels, CSF GNA levels remained stable within each cohort over time. Notably, Ngly1 +/− rats showed modestly higher CSF GNA levels than Ngly1 +/+ controls at all time points. The Neuroinflammatory Markers IBA-1 and GFAP Exhibit Regional Specific Elevations in Ngly1 −/− rat brain Brain tissues from Ngly1 +/+ (N = 2), Ngly1 +/− (N = 2) that were terminated at 12 months of age, and Ngly1 −/− (N = 15) rats that died or were humanly euthanized at 12 months of age were analyzed for neuroinflammation using the astrocytic marker GFAP (Glial Fibrillary Acidic Protein) and the microglial marker IBA-1. Due to limited sample sizes for the Ngly1 +/+ and Ngly1 +/− groups (N = 2 for each group) and a lack of detectable phenotypic or physiological differences, these two genotypes (4 rats in total) were combined into one group ( Ngly1 +/+ + Ngly1 +/− ) for comparison with the Ngly1 −/− rat brain tissues given minimal differences were observed between Ngly1 +/+ and Ngly1 +/− rats in other assessments. The analysis of the GFAP and IBA-1 staining included (1) the percentage of GFAP- or IBA-1–positive cells relative to the total number of cells (DAPI-positive cells) in each brain region, and (2) the average immunofluorescence intensity of GFAP or IBA-1 staining (Fig. 6 ). Brain tissue from Ngly1 −/− rats exhibited a significantly higher positive percentage of IBA-1 positive cells than brain tissue from Ngly1 +/+ and Ngly1 +/− rats in several brain regions: cerebellum (1.86 ± 0.12% vs. 1.18 ± 0.15%, p = 0.014, t-test), cerebral cortex: (4.56 ± 0.16% vs 3.82 ± 0.21%, p = 0.032, t-test), and medulla oblongata (7.32 ± 0.83% vs 4.60 ± 0.68%, p = 0.032, t-test) (Fig. 6 A). Ngly1 −/− rats also exhibited a trend toward higher IBA-1-positive cell percentage (4.71 ± 0.23%) compared to Ngly1 +/+ + Ngly1 +/− rats (3.87 ± 0.47%; p = 0.093) in the amygdala. The IBA-1 staining intensity in Ngly1 −/− rat brain tissue was also significantly higher than in Ngly1 +/+ and Ngly1 +/− rats in the following brain regions: caudate/putamen (66.28 ± 1.79 vs 54.03 ± 3.64 units, p = 0.019, t-test), nucleus accumbens (66.59 ± 2.10 vs 55.85 ± 2.56 units, p = 0.015, t-test)amygdala (69.08 ± 1.95 vs 59.10 ± 1.74 units, p = 0.061, t-test), cerebral cortex (67.24 ± 1.32 vs 59.74 ± 2.92 units, p = 0.051, t-test), and thalamus (66.55 ± 2.04 vs 59.31 ± 2.04 units, p = 0.080) (Fig. 6 B). Ngly1 −/− rat brains tissue showed significantly higher positive percentage of GFAP positive cells than Ngly1 +/+ and Ngly1 +/− rats in the amygdala (23.06 ± 0.61% vs 11.66 ± 3.35%, p = 0.038, t-test), but this was not observed in other brain tissue regions ( Supplemental Fig. 2 ). These data indicate significant, region-specific neuroinflammation in Ngly1 −/− rat brain tissue, with clear statistical differences in certain areas and additional regions exhibiting trends. This suggests broad inflammatory disease pathology, potentially contributing to neuropathological outcomes observed in NGLY1 Deficiency. Hematological and Histopathological Analysis in Ngly1 −/− rat A trend of increased large unstained cells (LUC) and monocytes was observed in Ngly1 −/− rat hematology compared to Ngly1 +/+ and Ngly1 +/− rats ( Supplemental Fig. 3 ). All other hematology measurements in Ngly1 −/− rats were similar to Ngly1 +/+ and Ngly1 +/− rats. Histopathological analysis of Ngly1 ⁻ / ⁻ central and peripheral nervous tissues revealed several abnormalities not seen in Ngly1 +/+ or Ngly1 +/− rats. These included CNS mineralization in certain brain regions, eosinophilic inclusions within the medulla oblongata, spinal cord gray matter, and dorsal root ganglia (DRG), as well as signs of neurodegeneration in DRG and axonal degeneration in spinal cord tracts, DRG, and sciatic nerve (data not shown). Discussion The assessment of aging in Ngly1 −/− rats in the reported study provides a comprehensive characterization of the disease over time that mirrors the progressive neurodegeneration observed in patients with NGLY1 Deficiency. Specifically, the pronounced motor deficits, premature mortality, and neuroinflammatory markers observed in these rats align with what is known about the phenotype progression and neurodegeneration in patients [ 19 ], underscoring the model's translational relevance. In addition to the previously described early postnatal mortality observed prior to weaning at PND 21 (Asahina 2020), we observed another major mortality event at 8.5 ~ 10 months of age that has not been previously reported. This data is consistent with reported deaths in patients during the course of an NGLY1 Natural History Study [ 7 ] and shortened lifespan (Grace Science Foundation) and underscores the need for early intervention to prevent shortened lifespan in patients with NGLY1 Deficiency. The study's finding that aging Ngly1 −/− rats continue to have elevated GNA levels in CSF and plasma, further validates GNA as a robust and sustained biomarker for NGLY1 Deficiency [ 16 ]. Phenotypic assessments in this study began at approximately 6 months of age. Based on the results, Ngly1 −/− rats appear to have already reached near-maximal motor impairment at this age. For example, rotarod latency was close to zero at 6.9 months (Fig. 3 ), consistent with prior reports of progressive decline [ 14 ]. Although rearing activity remained stable between 6.9 and 10.4 months, it was already significantly reduced compared to Ngly1 +/+ and Ngly1 +/− littermates at that time point (Fig. 4 ). In contrast, the significant reduction in basic movements observed after 6 months was not present in younger animals, suggesting ongoing functional deterioration beyond early adulthood [ 14 ]. Moreover, the neuroinflammation observed in Ngly1 −/− rats exhibiting premature mortality or clinical deterioration that required human euthanasia, characterized by increased expression of IBA-1 and GFAP in specific brain regions, suggests an inflammatory component to the disease's pathology. This is consistent with previous reports of IBA-1 and ubiquitin pathology in younger Ngly1 −/− rats [ 13 ] and with postmortem findings from NGLY1-deficient patients showing neuronal inclusions and Purkinje cell loss [ 19 ]. The neuroinflammation in rat cortex and the observed DRG pathology is also consistent with human autopsy findings of eosinophilic cytoplasmic inclusions in the cortex and DRG [ 19 ]. Interestingly, the neuroinflammation was mostly restricted to the thalamus in younger Ngly1 −/− rats [ 13 ] but seems to progress and was found to be more widespread acorss multiple brain regions in the older rats observed in this study. The neuroinflammation and neurodegeneration suggest a potential causal link to the progressive motor and neurological decline observed in both Ngly1 −/− rat and human NGLY1 Deficiency patients. Such inflammatory responses are common in other neurodegenerative disorders, indicating the potential for shared pathogenic mechanisms that may be therapeutically targeted [ 20 , 21 ]. In conclusion, the Ngly1 −/− rat model, when studied longitudinally through its lifespan, effectively recapitulates key aspects of human NGLY1 Deficiency, offering a valuable model of disease that can be used to evaluate potential therapeutic interventions. The integration of molecular, biochemical, and histopathological analyses in this model enhances the understanding of the disease progression and highlights the need for therapeutic interventions. Material and Methods Animals The Sprague-Dawley Ngly1 −/− rat was previously generated by deleting approximately 2.6 kb in exon 11 and exon 12 and a 3′ flanking region of the Ngly1 gene via CRISPR-Cas9 gene editing technology (Asahina, 2020). Animal care procedures and experiments conformed to the United States Department of Agriculture (USDA) Animal Welfare Act (Code of Federal Regulations, Title 9 [9 CFR], Parts 1, 2, and 3) and are under the strict oversight of the Institutional Animal Care and Use Committee (IACUC), Ethics Committee (EC) and Animal Welfare Body (AWB) at Charles River Labs. Rats were pair-housed where possible. Animals were separated during designated procedures/activities and as required for monitoring and/or health purposes, as deemed appropriate by the principal investigator and/or clinical veterinarian. Animals were housed in solid-bottom cages with nonaromatic bedding. Fluorescent lighting was provided via an automatic timer for approximately 12 h per day. The basal diet was block Lab Diet Certified Rodent Diet #5002, PMI Nutrition International. Tap water was offered ad libitum to all animals via an automatic water system. Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. Treatment of the animal(s) for minor injuries or ailments was approved when such treatment did not affect fulfillment of the study objectives. Mortality/Cageside Observations, Clinical Observations, and Humane Euthanasia Animals were observed within their cage twice daily for morbidity, mortality, injury, and availability of food and water. Any animals in poor health were identified for further monitoring and possible euthanasia. Poor health was determined by the observation that the animal was in overt pain/distress or appeared moribund and was beyond the point where recovery appeared reasonable. Animals in poor health were euthanized for humane reasons in accordance with the American Veterinary Medical Association (AVMA) Guidelines on Euthanasia. Animals were removed from the cage for detailed clinical observation monthly. Observations will include evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, respiratory and circulatory effects, autonomic effects such as salivation, nervous system effects including tremors, convulsions, reactivity to handling, and unusual behavior. Animal genotyping A PCR genotyping assay that targeted the Ngly1 gene locus was used to differentiate between Ngly1 +/+ , Ngly1 +/− , and Ngly1 −/− rats. All rats were produced as described in Mueller et al [16]. Rotarod, locomotor, and FOB testing The accelerating rotarod test is designed to measure balance, coordination, physical condition, and motor learning. Rats underwent rotarod testing using the San Diego Rotor-Rod Rod System. Conditioning sessions were performed on the morning of or a day prior to the actual testing and involved the animal being placed on the rotarod set to a constant speed of − 4 rpm (the minus sign indicates that the rod is moving in a direction that causes the rat to ambulate away from the observer) for 4 min. During the actual test, animals were placed on a rotating rod programmed to accelerate from 4 to 40 rpm at a rate of 0.15 revolutions per second over the course of 4 min. For animals staying on the rotarod for the entire 4-min period, a time of 240 s was recorded as the fall time. The elapsed time from the start of the trial to the animal’s fall was recorded digitally by the instrument. Each animal was evaluated in three trials with at least 15 min between trials. The average of all three trials is reported for each animal. Locomotor testing was performed by placing the animals into a Hamilton-Kinder enclosure. The duration of monitoring of rearing activity was 15 min. General linear model with genotypes, time point, and route of administration as effects was used for statistical analysis comparing the averaged fall time (rotarod) and rearing activity for rotarod and locomotor, respectively. FOB testing was conducted on all animals without knowledge on the part of the testers of the treatment status of each animal. Each animal was observed for a minimum of 3 min in a black Plexiglas open-field observation box. Parameters evaluated were based on those outlined by Moser and coworkers [22, 23]. Pathological analysis Brains and other tissues for histology were preserved in 10% neutral buffered formalin (NBF) for 24–48 hours and transferred to 70% ethanol for up to an additional 72 hours prior to processing to paraffin. Slices (2µm) were generated from prepared tissue blocks and mounted on slides for histopathological evaluation or immunohistochemistry staining by a pathologist. Quantitative GNA measurements Rat terminal CSF was collected by intracisternal magna (ICM) puncture, drawn with a syringe, frozen, and later analyzed. Plasma samples were isolated from blood taken via the sublingual vein (in-life collections) or cardiac puncture (terminal collection). Hemolysis was noted if present. Tissue samples were taken upon sacrifice without perfusion. Samples were frozen, dissected to 20- to 50-mg pieces while frozen, homogenized, and analyzed. Prepared rat tissue samples were analyzed as previously described [15]. Briefly, samples were homogenized in phosphate buffered saline (PBS), mixed with internal standard and acetonitrile, and centrifuged. An aliquot of each supernatant was separated via high-pressure liquid chromatography (HPLC) (Shimadzu VP Series 10 System, Shimadzu Corporation, Japan) and analyzed via tandem mass spectrometry (MS/MS) (Applied Biosystems/MDS SciEx API 4000, Danaher Corporation, Washington DC). Detection and accuracy were assessed in surrogate matrices (PBS + bovine serum albumin (BSA), charcoal stripped serum). Each surrogate matrix was spiked with 30 or 300 ng/mL of GNA, processed, and analyzed to determine recovery and accuracy. Statistical analysis Analyses were performed using R or GraphPad Prism version 10.3.1 for Mac (GraphPad Software, www.graphpad.com). Statistical significance of paired or multiple groups was evaluated by appropriate methods indicated in each figure legend. p 0.05; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001. Group data were represented by mean ± SEM. Declarations Availability of data and materials Data and scripts used for analysis will be available upon request Funding Statement & Acknowledgments The studies were funded by Grace Science Foundation. Author contributions L.Z., W.M., S.D., and B.S. designed the animal studies. L.Z., W.M., S.D., and B.S. managed the animal studies. L.Z. and W.M. analyzed the animal studies. L.Z., W.M., S.D., B.S. wrote the paper. Declaration of Interests All authors are employed by Grace Science, LLC. All authors are members of Grace Science Foundation Work was funded by Grace Science Foundation. References Tadashi Suzuki, Haruhiko Fujihira, NGLY1: A fascinating, multifunctional molecule, Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1868, Issue 2, 2024, 130379, ISSN 0304-4165. Manole A, Wong T, Rhee A, Novak S, Chin SM, Tsimring K, Paucar A, Williams A, Newmeyer TF, Schafer ST, Rosh I, Kaushik S, Hoffman R, Chen S, Wang G, Snyder M, Cuervo AM, Andrade L, Manor U, Lee K, Jones JR, Stern S, Marchetto MC, Gage FH. NGLY1 mutations cause protein aggregation in human neurons. Cell Rep. 2023 Dec 26;42(12):113466. doi: 10.1016/j.celrep.2023.113466. Epub 2023 Nov 30. PMID: 38039131; PMCID: PMC10826878. 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Tomlin FM, Gerling-Driessen UIM, Liu YC, Flynn RA, Vangala JR, Lentz CS, Clauder-Muenster S, Jakob P, Mueller WF, Ordoñez-Rueda D, Paulsen M, Matsui N, Foley D, Rafalko A, Suzuki T, Bogyo M, Steinmetz LM, Radhakrishnan SK, Bertozzi CR. Inhibition of NGLY1 Inactivates the Transcription Factor Nrf1 and Potentiates Proteasome Inhibitor Cytotoxicity. ACS Cent Sci. 2017 Nov 22;3(11):1143-1155. doi: 10.1021/acscentsci.7b00224. Epub 2017 Oct 25. PMID: 29202016; PMCID: PMC5704294. Levy RJ, Frater CH, Gallentine WB, Phillips JM, Ruzhnikov MR. Delineating the epilepsy phenotype of NGLY1 deficiency. J Inherit Metab Dis. 2022 May;45(3):571-583. doi: 10.1002/jimd.12494. Epub 2022 Mar 11. PMID: 35243670. Tong S, Ventola P, Frater CH, Klotz J, Phillips JM, Muppidi S, Dwight SS, Mueller WF, Beahm BJ, Wilsey M, Lee KJ. NGLY1 deficiency: a prospective natural history study. Hum Mol Genet. 2023 Sep 5;32(18):2787-2796. doi: 10.1093/hmg/ddad106. PMID: 37379343; PMCID: PMC10481101. Panneman DM, Wortmann SB, Haaxma CA, van Hasselt PM, Wolf NI, Hendriks Y, Küsters B, van Emst-de Vries S, van de Westerlo E, Koopman WJH, Wintjes L, van den Brandt F, de Vries M, Lefeber DJ, Smeitink JAM, Rodenburg RJ. Variants in NGLY1 lead to intellectual disability, myoclonus epilepsy, sensorimotor axonal polyneuropathy and mitochondrial dysfunction. Clin Genet. 2020 Apr;97(4):556-566. doi: 10.1111/cge.13706. Epub 2020 Jan 30. PMID: 31957011; PMCID: PMC7078978. Stanclift CR, Dwight SS, Lee K, Eijkenboom QL, Wilsey M, Wilsey K, Kobayashi ES, Tong S, Bainbridge MN. NGLY1 deficiency: estimated incidence, clinical features, and genotypic spectrum from the NGLY1 Registry. Orphanet J Rare Dis. 2022 Dec 17;17(1):440. doi: 10.1186/s13023-022-02592-3. PMID: 36528660; PMCID: PMC9759919. Lam C, Wolfe L, Need A, Shashi V, Enns G. NGLY1-Related Congenital Disorder of Deglycosylation. 2018 Feb 8. In: Adam MP, Feldman J, Mirzaa GM, Pagon RA, Wallace SE, Amemiya A, editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993–2025. PMID: 29419975. Enns GM, Shashi V, Bainbridge M, Gambello MJ, Zahir FR, Bast T, Crimian R, Schoch K, Platt J, Cox R, Bernstein JA, Scavina M, Walter RS, Bibb A, Jones M, Hegde M, Graham BH, Need AC, Oviedo A, Schaaf CP, Boyle S, Butte AJ, Chen R, Chen R, Clark MJ, Haraksingh R; FORGE Canada Consortium; Cowan TM, He P, Langlois S, Zoghbi HY, Snyder M, Gibbs RA, Freeze HH, Goldstein DB. Mutations in NGLY1 cause an inherited disorder of the endoplasmic reticulum-associated degradation pathway. Genet Med. 2014 Oct;16(10):751-8. doi: 10.1038/gim.2014.22. Epub 2014 Mar 20. Erratum in: Genet Med. 2014 Jul;16(7):568. Chen, Rui [added]. PMID: 24651605; PMCID: PMC4243708. Fujihira H, Masahara-Negishi Y, Tamura M, Huang C, Harada Y, Wakana S, Takakura D, Kawasaki N, Taniguchi N, Kondoh G, Yamashita T, Funakoshi Y, Suzuki T. Lethality of mice bearing a knockout of the Ngly1-gene is partially rescued by the additional deletion of the Engase gene. PLoS Genet. 2017 Apr 20;13(4):e1006696. doi: 10.1371/journal.pgen.1006696. PMID: 28426790; PMCID: PMC5398483. Asahina M, Fujinawa R, Nakamura S, Yokoyama K, Tozawa R, Suzuki T. Ngly1 -/- rats develop neurodegenerative phenotypes and pathological abnormalities in their peripheral and central nervous systems. Hum Mol Genet. 2020 Jun 27;29(10):1635-1647. doi: 10.1093/hmg/ddaa059. PMID: 32259258; PMCID: PMC7322575. Zhu L, Tan B, Dwight SS, Beahm B, Wilsey M, Crawford BE, Schweighardt B, Cook JW, Wechsler T, Mueller WF. AAV9-NGLY1 gene replacement therapy improves phenotypic and biomarker endpoints in a rat model of NGLY1 Deficiency. Mol Ther Methods Clin Dev. 2022 Oct 3;27:259-271. doi: 10.1016/j.omtm.2022.09.015. PMID: 36320418; PMCID: PMC9593239. Asahina M, Fujinawa R, Fujihira H, Masahara-Negishi Y, Andou T, Tozawa R, Suzuki T. JF1/B6F1 Ngly1-/- mouse as an isogenic animal model of NGLY1 deficiency. Proc Jpn Acad Ser B Phys Biol Sci. 2021;97(2):89-102. doi: 10.2183/pjab.97.005. PMID: 33563880; PMCID: PMC7897899. Mueller WF, Zhu L, Tan B, Dwight S, Beahm B, Wilsey M, Wechsler T, Mak J, Cowan T, Pritchett J, Taylor E, Crawford BE. GlcNAc-Asn is a biomarker for NGLY1 deficiency. J Biochem. 2022 Feb 21;171(2):177-186. doi: 10.1093/jb/mvab111. Erratum in: J Biochem. 2022 Mar 31;171(4):469. doi: 10.1093/jb/mvac016. PMID: 34697629; PMCID: PMC8863169. Crawley JN. Behavioral phenotyping of rodents. Comp. Med. 2003;53:140–146. Curzon P. 2009. The Behavioral Assessment of Sensorimotor. Stuut T, Popescu O, Oviedo A. N-Glycanase 1 Deficiency Is a Rare Cause of Pediatric Neurodegeneration With Neuronal Inclusions and Liver Steatosis. Cureus. 2021 Oct 29;13(10):e19126. doi: 10.7759/cureus.19126. PMID: 34858763; PMCID: PMC8614178. Amor S, Peferoen LA, Vogel DY, Breur M, van der Valk P, Baker D, van Noort JM. Inflammation in neurodegenerative diseases--an update. Immunology. 2014 Jun;142(2):151-66. doi: 10.1111/imm.12233. PMID: 24329535; PMCID: PMC4008224. Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH. Mechanisms underlying inflammation in neurodegeneration. Cell. 2010 Mar 19;140(6):918-34. doi: 10.1016/j.cell.2010.02.016. PMID: 20303880; PMCID: PMC2873093. Moser VC, McCormick JP, Creason JP, MacPhail RC. Comparison of chlordimeform and carbaryl using a functional observational battery. Fundam Appl Toxicol. 1988 Aug;11(2):189-206. doi: 10.1016/0272-0590(88)90144-3. PMID: 3146518. Moser VC. Rat strain- and gender-related differences in neurobehavioral screening: acute trimethyltin neurotoxicity. J Toxicol Environ Health. 1996 Apr 19;47(6):567-86. doi: 10.1080/009841096161546. PMID: 8614024. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7014298","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":511552048,"identity":"0ad4c16f-e199-4791-99fa-e26df9e38a5b","order_by":0,"name":"Lei Zhu","email":"","orcid":"","institution":"Grace Science Foundation","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Zhu","suffix":""},{"id":511552049,"identity":"0bd033ce-0378-4a15-a3e1-d309f1039f34","order_by":1,"name":"Selina Dwight","email":"","orcid":"","institution":"Grace Science Foundation","correspondingAuthor":false,"prefix":"","firstName":"Selina","middleName":"","lastName":"Dwight","suffix":""},{"id":511552050,"identity":"418edda1-6f77-47b4-9c7b-57c317868924","order_by":2,"name":"William Mueller","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYDACZubGAx9gnATitDA2HJxBmhYGxobDPCS5y+A4UIvNr3vy/O3tDz88qLgjz8/AfOzjF3xaDgO15PYVG844c8ZYIuHMM8OZDWzJs2XwaDEDa+lJYNwgkcPGkNh2OMHgAI8xswQhLZY9CfYb5J8/Y0j8dzjBnigtDD8SEjdIMJgxJDYAbWHgMWb8gEeLPVDLwd6GhOQZZ3KAfjl22HDGYbZkZjw6GCT7Dx988ONPgm1/+/GHH3/UHAYGXfNhxh/49IAAYxsyD2gFM+GY+oNuBkFbRsEoGAWjYCQBANJCUwiCcF/gAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-9134-7648","institution":"Grace Science Foundation","correspondingAuthor":true,"prefix":"","firstName":"William","middleName":"","lastName":"Mueller","suffix":""},{"id":511552051,"identity":"815cdb17-f537-4e4e-b6d2-71f7f455a744","order_by":3,"name":"Becky Schweighardt","email":"","orcid":"","institution":"Grace Science Foundation","correspondingAuthor":false,"prefix":"","firstName":"Becky","middleName":"","lastName":"Schweighardt","suffix":""}],"badges":[],"createdAt":"2025-06-30 22:02:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7014298/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7014298/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13023-026-04261-1","type":"published","date":"2026-02-20T15:57:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91305815,"identity":"200a01a2-e8fb-4f93-83e9-4fdf199b2fd0","added_by":"auto","created_at":"2025-09-15 06:30:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1744775,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBody Weight Growth Curve of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNgly1\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e+/+\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNgly1\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e+/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e,\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNgly1\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e rats Over Study Course.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBody weight was recorded monthly from 6 months of age until humane euthanasia or study termination for \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (N=10), \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e (N=10), and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (N=18) rats. Data are presented as mean ± SEM for each group at each recorded time point. **P\u0026lt;0.01, Linear mixed model.\u003c/p\u003e","description":"","filename":"Figure1AdjustedBodyWeightPlotMonthsLinear.png","url":"https://assets-eu.researchsquare.com/files/rs-7014298/v1/5d43b7928a5ddfeff8b45bd0.png"},{"id":91305814,"identity":"1e5106e7-e072-439b-8e65-18da7f114913","added_by":"auto","created_at":"2025-09-15 06:30:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1357305,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSurvival Curve of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNgly1\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e+/+\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e , \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNgly1\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e+/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e,\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNgly1\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA Kaplan–Meier survival curve for the three rat study cohorts was plotted from PND 21 (weaning) to 12 months. The number of rats for each genotype is indicated in the figure legend. All remaining \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e rats (along with 2 \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and 2 \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e rats as controls) were humane euthanized at 12 months of age due to deteriorating health conditions and their tissues preserved.\u003c/p\u003e","description":"","filename":"Figure2survivalcurvebygenotype.png","url":"https://assets-eu.researchsquare.com/files/rs-7014298/v1/b2b4e9f0818a870b9048f785.png"},{"id":91307022,"identity":"c1c03a28-2ee1-45ec-9807-0846b5e11ec8","added_by":"auto","created_at":"2025-09-15 06:38:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1667055,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRotarod Performance of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNgly1\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e+/+\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNgly1\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e+/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e,\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNgly1\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e rats Over Study Course\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRats were tested for latency to fall on rotating rotarod at 6.9, 10.4, and 16.6 months of age (all \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e rats were terminated by 12 months of age so were only tested at the first two time points). A single conditioning rotarod session was performed prior to the actual testing. Each animal was evaluated for its ability to remain on the accelerating rotarod (latency to fall) in 3 trials with at least 15 minutes between trials. The average of all 3 trials is reported for each animal, and the median time per group is shown as a line. Statistical significance was evaluated using Kruskal-Wallis test followed by Dunn’s multiple comparison. **** p\u0026lt;0.0001; ns, not significant.\u003c/p\u003e","description":"","filename":"Figure3Rotarod.png","url":"https://assets-eu.researchsquare.com/files/rs-7014298/v1/62dffb947bfdd9554d334a33.png"},{"id":91307023,"identity":"26a21ea5-b3c6-445b-aa13-289f04a921cb","added_by":"auto","created_at":"2025-09-15 06:38:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1069602,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLocomotor open field test\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eof \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNgly1\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e+/+\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNgly1\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e+/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e,\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNgly1\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e rats Over Study Course\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLocomotor testing at 6.9, 9, and 10.8 months of age for each of the 3 rat cohorts was performed by placing the animals into a Hamilton-Kinder enclosure and monitoring basic movement and rearing activity for 15 minutes. \u003cem\u003eNgly1\u003c/em\u003e⁻\u003csup\u003e/\u003c/sup\u003e⁻ rats were not tested at 10.8 months of age due to premature mortality or deteriorating health in this cohort. The total basic movements (A) and the total number of rearing events (B) per animal that completed the full 15-minutes sessions were totaled and plotted; the median rearing number per group is indicated as a line within the box plot. Statistics: generalized linear model.\u003c/p\u003e","description":"","filename":"Figure4Locomotor.png","url":"https://assets-eu.researchsquare.com/files/rs-7014298/v1/2835d45300b956cc0a6a16c9.png"},{"id":91305820,"identity":"3b8bdc27-f5fb-4503-b4ca-8392c6ec8346","added_by":"auto","created_at":"2025-09-15 06:30:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1519101,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLongitudinal GNA levels of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNgly1\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e+/+\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNgly1\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e+/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e,\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNgly1\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e rats in CSF and Plasma over time. \u003c/strong\u003eGNA biomarker levels were evaluated for CSF and plasma samples at multiple time points for \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e rats, as indicated in the graphs above; for \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e rats, CSF and plasma samples were evaluated only at the first three time points. Straight lines are linear regression of the levels for each genotype. **** P\u0026lt;0.0001, linear mixed effect model.\u003c/p\u003e","description":"","filename":"Figure5GNA.png","url":"https://assets-eu.researchsquare.com/files/rs-7014298/v1/a7af708cf79b86b2389bd6e3.png"},{"id":91307026,"identity":"cb2731b8-d5c3-43d5-9b5a-9cd4ccd1522e","added_by":"auto","created_at":"2025-09-15 06:38:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1424690,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIBA-1 percentage and intensity in different brain regions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue slices from rat brains were fixed, paraffin embedded, sliced, and immunostained with anti-IBA-1 antibodies.The slides were then scanned and analyzed using imaging software to quantify the number of IBA-1-positive cells and to calculate the percentage of cells with detectable IBA-1 expression by normalizing to total cell count (DAPI). Each dot represents the average of 8 slices per brain. Data are presented as mean box plots, where the box represents the interquartile range (25th–75th percentile), the line indicates the mean, whiskers show the minimum and maximum values, and outliers (if any) are plotted individually. Statistical significance was calculated using unpaired two-tailedt-test.\u003c/p\u003e","description":"","filename":"Figure6IBA1.png","url":"https://assets-eu.researchsquare.com/files/rs-7014298/v1/bb2291884546765a5af6b650.png"},{"id":103251073,"identity":"b776516e-0a9c-4954-9b0b-832d472a6656","added_by":"auto","created_at":"2026-02-23 16:03:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9843140,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7014298/v1/11e46566-68db-43a8-99e2-d218ff9c6124.pdf"},{"id":91305836,"identity":"6b150071-a4c7-410e-a595-d0d59359fe7e","added_by":"auto","created_at":"2025-09-15 06:30:16","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":579280,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7014298/v1/b10c8b2be72e3cb9c83c7806.pdf"}],"financialInterests":"","formattedTitle":"Progressive Neurodegeneration, Motor Decline, and Premature Mortality in Aging Ngly1 Deficient Rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNGLY1 Deficiency (OMIM #615273) is an ultra-rare autosomal recessive genetic disease caused by biallelic mutations in the \u003cem\u003eNGLY1\u003c/em\u003e gene. \u003cem\u003eNGLY1\u003c/em\u003e encodes N-glycanase 1, a conserved cytosolic enzyme that removes N-linked glycans from misfolded glycoproteins during endoplasmic reticulum-associated degradation (ERAD) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Loss of NGLY1 disrupts this critical protein quality-control pathway, leading to accumulation of undegraded glycoproteins and widespread cellular stress [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Clinically, NGLY1 Deficiency presents as a suite of complex neurodevelopmental phenotypes with global developmental delay and/or intellectual disability, a hyperkinetic movement disorder, transient elevation of liver transaminases, (hypo)alacrima (insufficient or absent tear production), and a chronic diffuse sensorimotor neuropathy that affects the nerves in a length-dependent manner. As children grow, a characteristic hyperkinetic movement disorder (chorea/dystonia) emerges frequently alongside epilepsy [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Peripheral neuropathy develops progressively, often manifesting as areflexia and distal weakness, and other musculoskeletal complications such as scoliosis and contractures can arise [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Notably, while early mortality was not prominent in initial case studies [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], recent longitudinal data suggest a limited lifespan in NGLY1 Deficiency patients, with a mean age of death of 13 (reported in 2022) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], recently increasing to 14.6 years [unpublished data, Grace Science Foundation]. Together, these observations suggest that NGLY1 Deficiency results in progressive deterioration and early mortality in patients.\u003c/p\u003e\u003cp\u003eAnimal models are crucial for understanding the disease mechanism and testing therapies, yet modeling NGLY1 Deficiency has been challenging. \u003cem\u003eNgly1\u003c/em\u003e-knockout mice in a C57BL/6 background exhibit embryonic lethality [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and thus preclude postnatal studies. Given the limitations in mice, an \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rat model was developed to enable postnatal and longitudinal investigations [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This hurdle was later partially overcome by using a mixed (JF1/B6) genetic background [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This rat model showed high early mortality (70%) within the first three weeks after birth, stabilizing after weaning (21 days), and recapitulated key clinical features of NGLY1 Deficiency, including failure to thrive, peripheral neuropathy, hyperkinetic movements, and delayed cognitive development [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHistopathological analyses in young adult \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats (up to ~\u0026thinsp;7 months old) revealed neurodegenerative changes such as neuron loss in the thalamus, astrogliosis, microgliosis, and peripheral nerve axonopathy [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. While providing valuable insights into disease pathology, these early studies were not designed to follow adult animals. Consequently, little is known about the trajectory of NGLY1 Deficiency into late adulthood in an animal model, including whether new phenotypes emerge or if the pathology worsens with age. This gap in understanding is relevant given that some NGLY1 Deficiency patients live beyond their teenage years despite the shortened mean lifespan associated with the disease [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. An aging study was therefore conducted in the \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rat model to inform late-stage disease mechanisms with the eventual aim of developing and evaluating interventions that slow disease progression and extend lifespan.\u003c/p\u003e\u003cp\u003eA longitudinal study of \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats was conducted to help characterize the course of NGLY1 Deficiency in aging animals. In this study, a cohort of homozygous \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats was followed from ~\u0026thinsp;6 months through 17\u0026ndash;18 months of age alongside \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e (heterozygous) and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (wild-type) littermate control cohorts. Throughout this period, animals underwent periodic evaluations of motor behavior (rotarod, open field locomotor activity and rearing, and functional observational battery), assessments of the NGLY1 Deficiency biomarker, GlcNAc-Asn (GNA in both plasma and CSF levels [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]), and clinical indicators of health (body weight, survival, cage-side/clinical observation, hematology, and clinical chemistry). At end of life, comprehensive histopathological examinations were performed on each cohort, focusing on the nervous system (brain, spinal cord, peripheral nerves) and other organs relevant to NGLY1 Deficiency disease pathology. Here, we report that NGLY1 Deficiency in a rat model of the disease leads to severe premature aging phenotypes, including significant motor decline and late-onset neuromuscular deterioration, as well as mid-life mortality, thereby mirroring the human disease in both severity and progression. Our findings extend the phenotypic characterization of \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals beyond early adulthood and underscore the utility of this rat model for studying the pathogenesis and treatment of NGLY1 Deficiency in its advanced stages.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eNgly1\u003c/b\u003e\u003csup\u003e\u003cb\u003e⁻/⁻\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eRats Exhibit Progressive Neurological Phenotypes, Reduced Body Weight, And Premature Mortality as They Age\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt study initiation (~\u0026thinsp;6 months of age), animals were assigned to one of three cohorts: wild-type (\u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e; N\u0026thinsp;=\u0026thinsp;10, 5 males/5 females), heterozygous (\u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e; N\u0026thinsp;=\u0026thinsp;10, 5 males/5 females), or homozygous (\u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e; N\u0026thinsp;=\u0026thinsp;18, 9 males/9 females). Additional animals were included in the \u003cem\u003eNgly1\u003c/em\u003e⁻/⁻ group to account for anticipated mortality-related loss of animal number. Male \u003cem\u003eNgly1\u003c/em\u003e⁻/⁻ rats weighed significantly less than \u003cem\u003eNgly1\u003c/em\u003e⁺/⁺ and \u003cem\u003eNgly1\u003c/em\u003e⁺/⁻ males throughout the study (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, linear mixed model), whereas female \u003cem\u003eNgly1\u003c/em\u003e⁻/⁻ body weights did not differ from controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSurvival was also assessed for each rat in the 3 cohorts. Based on prior observations in \u003cem\u003eNgly1\u003c/em\u003e⁻/⁻ rats, litter sizes were not counted during the first few postnatal days to avoid disturbing the nests and potentially increasing postnatal mortality. Due to this, overall survival in the postnatal period from time of birth could not be assessed. After genotyping at postnatal day (PND) 15 and weaning at PND 21, the survival was 100% in all genotypes until ~\u0026thinsp;8.5 months of age, at which point \u003cem\u003eNgly1\u003c/em\u003e⁻/⁻ rats exhibited a premature mortality that was not observed in either \u003cem\u003eNgly1\u003c/em\u003e⁺/⁺ or \u003cem\u003eNgly1\u003c/em\u003e⁺/⁻ cohorts. Eight of 18 \u003cem\u003eNgly1\u003c/em\u003e⁻/⁻ rats (44%) died or required humane euthanasia due to severe decline in cage side/clinical observations between PND 255 and 299 (~\u0026thinsp;8.5\u0026ndash;10 months of age). The remaining 10 \u003cem\u003eNgly1\u003c/em\u003e⁻/⁻ animals showed various signs of severe health deterioration and were humanely euthanized at 12 months and their tissues preserved.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNgly1\u003c/b\u003e\u003cb\u003e⁻/⁻ Rats Exhibit Severe and Persistent Motor Deficits in the Rotarod Assessment, s\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe rotarod test measures the latency for each rat to fall from a rotating rod and is a measure of motor function [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Rotarod performance was assessed at 6.9 and 10.4 months of age for \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats and was determined to be significantly impaired in \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats compared to both \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats. At ~\u0026thinsp;6.9 months of age, \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats exhibited a latency to fall of 8.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7 seconds, which was significantly shorter than \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (81.4\u0026thinsp;\u0026plusmn;\u0026thinsp;11.0 s, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.4\u0026times;10⁻⁴) and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e (103.2\u0026thinsp;\u0026plusmn;\u0026thinsp;18.6 s, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.0\u0026times;10⁻⁵) rats. Similarly, at 10.4 months, \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats maintained significantly lower performance (6.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 seconds) than \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats which sustained motor coordination with latencies of 80.4\u0026thinsp;\u0026plusmn;\u0026thinsp;10.9 and 81.1\u0026thinsp;\u0026plusmn;\u0026thinsp;11.6 seconds, respectively (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;4\u0026times;10⁻⁷). No significant differences in rotarod performance were observed between \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats at any tested time point, including at 16.6 months of age, indicating that the severe motor deficit is specific to complete loss of NGLY1. Data from \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats were not available for a comparison of rotarod performance at 16.6 months due to the premature mortality of some animals and the significant deterioration of the surviving animals.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNgly1\u003c/b\u003e\u003cb\u003e⁻/⁻ Rats Show Significantly Reduced Locomotor Activity and Rearing at Compared with\u003c/b\u003e \u003cb\u003eNgly1\u003c/b\u003e\u003csup\u003e\u003cb\u003e+/+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eNgly1\u003c/b\u003e\u003csup\u003e\u003cb\u003e+/\u0026minus;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eRats\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLocomotor activity was assessed for all 3 rat cohorts at 6.9 and 9 months of age in an open field test using a Hamilton-Kinder enclosure, where horizontal (basic movement) and vertical (rearing) movements were recorded via infrared beam breaks over a 15-minute session. In the open field test, basic movement and number of rearings were both significantly lower in \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats compared to \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats at both 6.9-month and 9-month time points. No significant difference in basic movements or number of rearings was observed between \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats at any of the tested time points (6.9, 9, and 10.8 months; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAt 6.9 months of age, \u003cem\u003eNgly1\u003c/em\u003e⁻/⁻ rats showed markedly lower basic movements (17,909.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1,193.5) compared to \u003cem\u003eNgly1\u003c/em\u003e⁺/⁺ (27,195.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2,463.6, p\u0026thinsp;=\u0026thinsp;0.0087) and \u003cem\u003eNgly1\u003c/em\u003e⁺/⁻ (26,579.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2,495.3, p\u0026thinsp;=\u0026thinsp;0.0194) rats. A similar pattern was observed in rearing activity, where \u003cem\u003eNgly1\u003c/em\u003e⁻/⁻ rats displayed a significantly lower number of rearings (204.4\u0026thinsp;\u0026plusmn;\u0026thinsp;25.2) than both \u003cem\u003eNgly1\u003c/em\u003e⁺/⁺ (748.6\u0026thinsp;\u0026plusmn;\u0026thinsp;122.0, p\u0026thinsp;=\u0026thinsp;0.0007) and \u003cem\u003eNgly1\u003c/em\u003e⁺/⁻ (813.9\u0026thinsp;\u0026plusmn;\u0026thinsp;173.3, p\u0026thinsp;=\u0026thinsp;0.0006) cohorts.\u003c/p\u003e\u003cp\u003eAt 9.0 months of age, \u003cem\u003eNgly1\u003c/em\u003e⁻/⁻ rats continued to exhibit significantly reduced basic movement (18,266.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1,431.5) compared to \u003cem\u003eNgly1\u003c/em\u003e⁺/⁺ (31,616.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2,285.0, p\u0026thinsp;=\u0026thinsp;0.0003) and \u003cem\u003eNgly1\u003c/em\u003e⁺/⁻ (28,474.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2,547.9, p\u0026thinsp;=\u0026thinsp;0.007) rats. As was also observed for rats 6.9 months of age, \u003cem\u003eNgly1\u003c/em\u003e⁻/⁻ rats at 9.0 months of age displayed a significantly lower number of rearings (176.8\u0026thinsp;\u0026plusmn;\u0026thinsp;27.8) than both \u003cem\u003eNgly1\u003c/em\u003e⁺/⁺ (1,013.2\u0026thinsp;\u0026plusmn;\u0026thinsp;138.0, p\u0026thinsp;=\u0026thinsp;5 \u0026times;10⁻\u003csup\u003e5\u003c/sup\u003e) and \u003cem\u003eNgly1\u003c/em\u003e⁺/⁻ (879.1\u0026thinsp;\u0026plusmn;\u0026thinsp;137.9, p\u0026thinsp;=\u0026thinsp;0.0003) rats.\u003c/p\u003e\u003cp\u003eData from \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats were not available for a comparison of basic movement and number of rearings at 10.8 months due to the premature mortality of some animals and the significant deterioration of the surviving animals, but \u003cem\u003eNgly1\u003c/em\u003e⁺/⁺ and \u003cem\u003eNgly1\u003c/em\u003e⁺/⁻ rats were tested at this time point. No significant differences in either basic movements or number of rearings were observed between \u003cem\u003eNgly1\u003c/em\u003e⁺/⁺ and \u003cem\u003eNgly1\u003c/em\u003e⁺/⁻ rats at any tested age, suggesting that loss of a single \u003cem\u003eNGLY1\u003c/em\u003e allele does not impair gross locomotor behavior as assessed in the locomotor open field test.\u003c/p\u003e\u003cp\u003eIn the Functional Observational Battery (FOB), a test of neurobehavioral behavior, \u003cem\u003eNgly1\u003c/em\u003e⁻\u003csup\u003e/\u003c/sup\u003e⁻ rats exhibited impairments in rearing (as assessed for 1-minute by a scorer), righting reflexes, and gait/mobility when compared to \u003cem\u003eNgly1\u003c/em\u003e⁺/⁺ and \u003cem\u003eNgly1\u003c/em\u003e⁺/⁻ rats at approximately 7 and 9 months of age (\u003cb\u003eSupplemental Fig.\u0026nbsp;1\u003c/b\u003e). No significant differences were observed in other FOB parameters (Data not shown). As with the locomotor open field test, there were no differences detected between \u003cem\u003eNgly1\u003c/em\u003e⁺\u003csup\u003e/\u003c/sup\u003e⁺ and \u003cem\u003eNgly1\u003c/em\u003e⁺\u003csup\u003e/\u003c/sup\u003e⁻ rats at any time point. (\u003cb\u003eSupplemental figure x, Data not shown\u003c/b\u003e)\u003c/p\u003e\u003cp\u003eBased on cage side clinical observations, \u003cem\u003eNgly1\u003c/em\u003e⁻\u003csup\u003e/\u003c/sup\u003e⁻ rats displayed a consistent neurological phenotype at study initiation (~\u0026thinsp;6 months of age) that was consistent with the previous characterization of this Ngly1 deficient rat model (Asahina et al 2020, Zhu et al 2022). Specifically, the \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats exhibited uncoordinated gait, hindlimb weakness (often asymmetric), bruxism, splayed limbs, head tilt, and labored breathing. Additional observations exclusive to \u003cem\u003eNgly1\u003c/em\u003e⁻\u003csup\u003e/\u003c/sup\u003e⁻ animals in this study included malocclusion, thin body condition, low head carriage, skin discoloration with scabbing, and dental discoloration. Overall, these phenotypes progressively worsened over time.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNgly1\u003c/b\u003e\u003cb\u003e⁻/⁻ rats Exhibit Consistently Elevated GNA levels in both plasma and CSF.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLongitudinal analyses of GNA levels in both cerebrospinal fluid (CSF) and plasma were conducted in \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003erats at the following time points: 6.2\u0026ndash;7.1 months, 8.7\u0026ndash;9.2 months, and 10.6\u0026ndash;11.3 months (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Throughout the observation period, plasma GNA levels were consistently higher in \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats compared with either \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e or \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats at each time point. Interestingly, GNA levels in the plasma of \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats demonstrated an age-dependent increase from 6.5 months of age to their death or humane euthanasia at 12 months of age, while plasma GNA levels in \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats remained at similar, low levels throughout the study.\u003c/p\u003e\u003cp\u003e\u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats also exhibited consistently elevated CSF GNA levels (252.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.3 ng/mL, 224.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.6 ng/mL, 336\u0026thinsp;\u0026plusmn;\u0026thinsp;73 ng/mL) compared to \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (27.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 ng/mL, 23.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 ng/mL, and 35.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 ng/mL)and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e (46.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 ng/mL, 43.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 ng/mL, 43.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 ng/mL) rats across all 3 evaluated time points. In contrast to plasma GNA levels, CSF GNA levels remained stable within each cohort over time. Notably, \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats showed modestly higher CSF GNA levels than \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e controls at all time points.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe Neuroinflammatory Markers IBA-1 and GFAP Exhibit Regional Specific Elevations in\u003c/b\u003e \u003cb\u003eNgly1\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003erat brain\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBrain tissues from \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (N\u0026thinsp;=\u0026thinsp;2), \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e (N\u0026thinsp;=\u0026thinsp;2) that were terminated at 12 months of age, and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e (N\u0026thinsp;=\u0026thinsp;15) rats that died or were humanly euthanized at 12 months of age were analyzed for neuroinflammation using the astrocytic marker GFAP (Glial Fibrillary Acidic Protein) and the microglial marker IBA-1. Due to limited sample sizes for the \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e groups (N\u0026thinsp;=\u0026thinsp;2 for each group) and a lack of detectable phenotypic or physiological differences, these two genotypes (4 rats in total) were combined into one group (\u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e + \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) for comparison with the \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rat brain tissues given minimal differences were observed between \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats in other assessments. The analysis of the GFAP and IBA-1 staining included (1) the percentage of GFAP- or IBA-1\u0026ndash;positive cells relative to the total number of cells (DAPI-positive cells) in each brain region, and (2) the average immunofluorescence intensity of GFAP or IBA-1 staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBrain tissue from \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats exhibited a significantly higher positive percentage of IBA-1 positive cells than brain tissue from \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats in several brain regions: cerebellum (1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12% vs. 1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15%, p\u0026thinsp;=\u0026thinsp;0.014, t-test), cerebral cortex: (4.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16% vs 3.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21%, p\u0026thinsp;=\u0026thinsp;0.032, t-test), and medulla oblongata (7.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83% vs 4.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68%, p\u0026thinsp;=\u0026thinsp;0.032, t-test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats also exhibited a trend toward higher IBA-1-positive cell percentage (4.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23%) compared to \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e + \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats (3.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47%; p\u0026thinsp;=\u0026thinsp;0.093) in the amygdala.\u003c/p\u003e\u003cp\u003eThe IBA-1 staining intensity in \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rat brain tissue was also significantly higher than in \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats in the following brain regions: caudate/putamen (66.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79 vs 54.03\u0026thinsp;\u0026plusmn;\u0026thinsp;3.64 units, p\u0026thinsp;=\u0026thinsp;0.019, t-test), nucleus accumbens (66.59\u0026thinsp;\u0026plusmn;\u0026thinsp;2.10 vs 55.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.56 units, p\u0026thinsp;=\u0026thinsp;0.015, t-test)amygdala (69.08\u0026thinsp;\u0026plusmn;\u0026thinsp;1.95 vs 59.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.74 units, p\u0026thinsp;=\u0026thinsp;0.061, t-test), cerebral cortex (67.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32 vs 59.74\u0026thinsp;\u0026plusmn;\u0026thinsp;2.92 units, p\u0026thinsp;=\u0026thinsp;0.051, t-test), and thalamus (66.55\u0026thinsp;\u0026plusmn;\u0026thinsp;2.04 vs 59.31\u0026thinsp;\u0026plusmn;\u0026thinsp;2.04 units, p\u0026thinsp;=\u0026thinsp;0.080) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rat brains tissue showed significantly higher positive percentage of GFAP positive cells than \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats in the amygdala (23.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61% vs 11.66\u0026thinsp;\u0026plusmn;\u0026thinsp;3.35%, p\u0026thinsp;=\u0026thinsp;0.038, t-test), but this was not observed in other brain tissue regions (\u003cb\u003eSupplemental Fig.\u0026nbsp;2\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eThese data indicate significant, region-specific neuroinflammation in \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rat brain tissue, with clear statistical differences in certain areas and additional regions exhibiting trends. This suggests broad inflammatory disease pathology, potentially contributing to neuropathological outcomes observed in NGLY1 Deficiency.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eHematological and Histopathological Analysis in\u003c/span\u003e \u003cspan type=\"BoldItalicUnderline\" class=\"BoldItalicUnderline\" name=\"Emphasis\"\u003eNgly1\u003c/span\u003e\u003csup\u003e\u003cspan type=\"BoldItalicUnderline\" class=\"BoldItalicUnderline\" name=\"Emphasis\"\u003e\u0026minus;/\u0026minus;\u003c/span\u003e\u003c/sup\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003erat\u003c/span\u003e\u003c/p\u003e\u003cp\u003eA trend of increased large unstained cells (LUC) and monocytes was observed in \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rat hematology compared to \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats (\u003cb\u003eSupplemental Fig.\u0026nbsp;3\u003c/b\u003e). All other hematology measurements in \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats were similar to \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats. Histopathological analysis of \u003cem\u003eNgly1\u003c/em\u003e⁻\u003csup\u003e/\u003c/sup\u003e⁻ central and peripheral nervous tissues revealed several abnormalities not seen in \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e or \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats. These included CNS mineralization in certain brain regions, eosinophilic inclusions within the medulla oblongata, spinal cord gray matter, and dorsal root ganglia (DRG), as well as signs of neurodegeneration in DRG and axonal degeneration in spinal cord tracts, DRG, and sciatic nerve (data not shown).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe assessment of aging in \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats in the reported study provides a comprehensive characterization of the disease over time that mirrors the progressive neurodegeneration observed in patients with NGLY1 Deficiency. Specifically, the pronounced motor deficits, premature mortality, and neuroinflammatory markers observed in these rats align with what is known about the phenotype progression and neurodegeneration in patients [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], underscoring the model's translational relevance. In addition to the previously described early postnatal mortality observed prior to weaning at PND 21 (Asahina 2020), we observed another major mortality event at 8.5\u0026thinsp;~\u0026thinsp;10 months of age that has not been previously reported. This data is consistent with reported deaths in patients during the course of an NGLY1 Natural History Study [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and shortened lifespan (Grace Science Foundation) and underscores the need for early intervention to prevent shortened lifespan in patients with NGLY1 Deficiency.\u003c/p\u003e\u003cp\u003eThe study's finding that aging \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats continue to have elevated GNA levels in CSF and plasma, further validates GNA as a robust and sustained biomarker for NGLY1 Deficiency [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePhenotypic assessments in this study began at approximately 6 months of age. Based on the results, \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats appear to have already reached near-maximal motor impairment at this age. For example, rotarod latency was close to zero at 6.9 months (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e), consistent with prior reports of progressive decline [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Although rearing activity remained stable between 6.9 and 10.4 months, it was already significantly reduced compared to \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e littermates at that time point (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In contrast, the significant reduction in basic movements observed after 6 months was not present in younger animals, suggesting ongoing functional deterioration beyond early adulthood [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMoreover, the neuroinflammation observed in \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats exhibiting premature mortality or clinical deterioration that required human euthanasia, characterized by increased expression of IBA-1 and GFAP in specific brain regions, suggests an inflammatory component to the disease's pathology. This is consistent with previous reports of IBA-1 and ubiquitin pathology in younger \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and with postmortem findings from NGLY1-deficient patients showing neuronal inclusions and Purkinje cell loss [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The neuroinflammation in rat cortex and the observed DRG pathology is also consistent with human autopsy findings of eosinophilic cytoplasmic inclusions in the cortex and DRG [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Interestingly, the neuroinflammation was mostly restricted to the thalamus in younger \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] but seems to progress and was found to be more widespread acorss multiple brain regions in the older rats observed in this study. The neuroinflammation and neurodegeneration suggest a potential causal link to the progressive motor and neurological decline observed in both \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rat and human NGLY1 Deficiency patients. Such inflammatory responses are common in other neurodegenerative disorders, indicating the potential for shared pathogenic mechanisms that may be therapeutically targeted [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn conclusion, the \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rat model, when studied longitudinally through its lifespan, effectively recapitulates key aspects of human NGLY1 Deficiency, offering a valuable model of disease that can be used to evaluate potential therapeutic interventions. The integration of molecular, biochemical, and histopathological analyses in this model enhances the understanding of the disease progression and highlights the need for therapeutic interventions.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Sprague-Dawley \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e rat was previously generated by deleting approximately 2.6 kb in exon 11 and exon 12 and a 3′ flanking region of the \u003cem\u003eNgly1\u003c/em\u003e gene via CRISPR-Cas9 gene editing technology (Asahina, 2020). Animal care procedures and experiments conformed to the United States Department of Agriculture (USDA) Animal Welfare Act (Code of Federal Regulations, Title 9 [9 CFR], Parts 1, 2, and 3) and are under the strict oversight of the Institutional Animal Care and Use Committee (IACUC), Ethics Committee (EC) and Animal Welfare Body (AWB) at Charles River Labs. Rats were pair-housed where possible. Animals were separated during designated procedures/activities and as required for monitoring and/or health purposes, as deemed appropriate by the principal investigator and/or clinical veterinarian. Animals were housed in solid-bottom cages with nonaromatic bedding. Fluorescent lighting was provided via an automatic timer for approximately 12 h per day. The basal diet was block Lab Diet Certified Rodent Diet #5002, PMI Nutrition International. Tap water was offered ad libitum to all animals via an automatic water system. Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. Treatment of the animal(s) for minor injuries or ailments was approved when such treatment did not affect fulfillment of the study objectives.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMortality/Cageside Observations, Clinical Observations, and Humane Euthanasia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimals were observed within their cage twice daily for morbidity, mortality, injury, and availability of food and water. Any animals in poor health were identified for further monitoring and possible euthanasia. Poor health was determined by the observation that the animal was in overt pain/distress or appeared moribund and was beyond the point where recovery appeared reasonable. Animals in poor health were euthanized for humane reasons in accordance with the American Veterinary Medical Association (AVMA) Guidelines on Euthanasia.\u003c/p\u003e\n\u003cp\u003eAnimals were removed from the cage for detailed clinical observation monthly. Observations will include evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, respiratory and circulatory effects, autonomic effects such as salivation, nervous system effects including tremors, convulsions, reactivity to handling, and unusual behavior.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal genotyping\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA PCR genotyping assay that targeted the \u003cem\u003eNgly1\u003c/em\u003e gene locus was used to differentiate between \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/−\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e rats. All rats were produced as described in Mueller et al [16].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRotarod, locomotor, and FOB testing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe accelerating rotarod test is designed to measure balance, coordination, physical condition, and motor learning. Rats underwent rotarod testing using the San Diego Rotor-Rod Rod System. Conditioning sessions were performed on the morning of or a day prior to the actual testing and involved the animal being placed on the rotarod set to a constant speed of − 4 rpm (the minus sign indicates that the rod is moving in a direction that causes the rat to ambulate away from the observer) for 4 min. During the actual test, animals were placed on a rotating rod programmed to accelerate from 4 to 40 rpm at a rate of 0.15 revolutions per second over the course of 4 min. For animals staying on the rotarod for the entire 4-min period, a time of 240 s was recorded as the fall time. The elapsed time from the start of the trial to the animal’s fall was recorded digitally by the instrument. Each animal was evaluated in three trials with at least 15 min between trials. The average of all three trials is reported for each animal.\u003c/p\u003e\n\u003cp\u003eLocomotor testing was performed by placing the animals into a Hamilton-Kinder enclosure. The duration of monitoring of rearing activity was 15 min. General linear model with genotypes, time point, and route of administration as effects was used for statistical analysis comparing the averaged fall time (rotarod) and rearing activity for rotarod and locomotor, respectively.\u003c/p\u003e\n\u003cp\u003eFOB testing was conducted on all animals without knowledge on the part of the testers of the treatment status of each animal. Each animal was observed for a minimum of 3 min in a black Plexiglas open-field observation box. Parameters evaluated were based on those outlined by Moser and coworkers [22, 23].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePathological analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBrains and other tissues for histology were preserved in 10% neutral buffered formalin (NBF) for 24–48 hours and transferred to 70% ethanol for up to an additional 72 hours prior to processing to paraffin. Slices (2µm) were generated from prepared tissue blocks and mounted on slides for histopathological evaluation or immunohistochemistry staining by a pathologist.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative GNA measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRat terminal CSF was collected by intracisternal magna (ICM) puncture, drawn with a syringe, frozen, and later analyzed. Plasma samples were isolated from blood taken via the sublingual vein (in-life collections) or cardiac puncture (terminal collection). Hemolysis was noted if present. Tissue samples were taken upon sacrifice without perfusion. Samples were frozen, dissected to 20- to 50-mg pieces while frozen, homogenized, and analyzed. Prepared rat tissue samples were analyzed as previously described [15]. Briefly, samples were homogenized in phosphate buffered saline (PBS), mixed with internal standard and acetonitrile, and centrifuged. An aliquot of each supernatant was separated via high-pressure liquid chromatography (HPLC) (Shimadzu VP Series 10 System, Shimadzu Corporation, Japan) and analyzed via tandem mass spectrometry (MS/MS) (Applied Biosystems/MDS SciEx API 4000, Danaher Corporation, Washington DC). Detection and accuracy were assessed in surrogate matrices (PBS + bovine serum albumin (BSA), charcoal stripped serum). Each surrogate matrix was spiked with 30 or 300 ng/mL of GNA, processed, and analyzed to determine recovery and accuracy.\u003c/p\u003e\n\u003cdiv id=\"Sec5\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eAnalyses were performed using R or GraphPad Prism version 10.3.1 for Mac (GraphPad Software, www.graphpad.com). Statistical significance of paired or multiple groups was evaluated by appropriate methods indicated in each figure legend. p \u0026lt; 0.05 was considered significant and is designated with an asterisk in all figures: ns, p \u0026gt; 0.05; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001. Group data were represented by mean ± SEM.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData and scripts used for analysis will be available upon request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement \u0026amp; Acknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe studies were funded by Grace Science Foundation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.Z., W.M., S.D., and B.S. designed the animal studies. L.Z., W.M., S.D., and B.S. managed the animal studies. L.Z. and W.M. analyzed the animal studies. \u0026nbsp;L.Z., W.M., S.D., B.S. wrote the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors are employed by Grace Science, LLC.\u003c/p\u003e\n\u003cp\u003eAll authors are members of Grace Science Foundation\u003c/p\u003e\n\u003cp\u003eWork was funded by Grace Science Foundation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTadashi Suzuki, Haruhiko Fujihira, NGLY1: A fascinating, multifunctional molecule, Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1868, Issue 2, 2024, 130379, ISSN 0304-4165.\u003c/li\u003e\n\u003cli\u003eManole A, Wong T, Rhee A, Novak S, Chin SM, Tsimring K, Paucar A, Williams A, Newmeyer TF, Schafer ST, Rosh I, Kaushik S, Hoffman R, Chen S, Wang G, Snyder M, Cuervo AM, Andrade L, Manor U, Lee K, Jones JR, Stern S, Marchetto MC, Gage FH. NGLY1 mutations cause protein aggregation in human neurons. Cell Rep. 2023 Dec 26;42(12):113466. doi: 10.1016/j.celrep.2023.113466. Epub 2023 Nov 30. PMID: 38039131; PMCID: PMC10826878.\u003c/li\u003e\n\u003cli\u003eWilliam F Mueller, Petra Jakob, Han Sun, Sandra Clauder-M\u0026uuml;nster, Sonja Ghidelli-Disse, Diana Ordonez, Markus Boesche, Marcus Bantscheff, Paul Collier, Bettina Haase, Vladimir Benes, Malte Paulsen, Peter Sehr, Joe Lewis, Gerard Drewes, Lars M Steinmetz, Loss of N-Glycanase 1 Alters Transcriptional and Translational Regulation in K562 Cell Lines, G3 Genes|Genomes|Genetics, Volume 10, Issue 5, 1 May 2020, Pages 1585\u0026ndash;1597\u003c/li\u003e\n\u003cli\u003eHuang C, Harada Y, Hosomi A, Masahara-Negishi Y, Seino J, Fujihira H, Funakoshi Y, Suzuki T, Dohmae N, Suzuki T. Endo-\u0026beta;-N-acetylglucosaminidase forms N-GlcNAc protein aggregates during ER-associated degradation in Ngly1-defective cells. Proc Natl Acad Sci U S A. 2015 Feb 3;112(5):1398-403. doi: 10.1073/pnas.1414593112. Epub 2015 Jan 20. PMID: 25605922; PMCID: PMC4321286.\u003c/li\u003e\n\u003cli\u003eTomlin FM, Gerling-Driessen UIM, Liu YC, Flynn RA, Vangala JR, Lentz CS, Clauder-Muenster S, Jakob P, Mueller WF, Ordo\u0026ntilde;ez-Rueda D, Paulsen M, Matsui N, Foley D, Rafalko A, Suzuki T, Bogyo M, Steinmetz LM, Radhakrishnan SK, Bertozzi CR. Inhibition of NGLY1 Inactivates the Transcription Factor Nrf1 and Potentiates Proteasome Inhibitor Cytotoxicity. ACS Cent Sci. 2017 Nov 22;3(11):1143-1155. doi: 10.1021/acscentsci.7b00224. Epub 2017 Oct 25. PMID: 29202016; PMCID: PMC5704294.\u003c/li\u003e\n\u003cli\u003eLevy RJ, Frater CH, Gallentine WB, Phillips JM, Ruzhnikov MR. Delineating the epilepsy phenotype of NGLY1 deficiency. J Inherit Metab Dis. 2022 May;45(3):571-583. doi: 10.1002/jimd.12494. Epub 2022 Mar 11. 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Cell. 2010 Mar 19;140(6):918-34. doi: 10.1016/j.cell.2010.02.016. PMID: 20303880; PMCID: PMC2873093.\u003c/li\u003e\n\u003cli\u003eMoser VC, McCormick JP, Creason JP, MacPhail RC. Comparison of chlordimeform and carbaryl using a functional observational battery. Fundam Appl Toxicol. 1988 Aug;11(2):189-206. doi: 10.1016/0272-0590(88)90144-3. PMID: 3146518.\u003c/li\u003e\n\u003cli\u003eMoser VC. Rat strain- and gender-related differences in neurobehavioral screening: acute trimethyltin neurotoxicity. J Toxicol Environ Health. 1996 Apr 19;47(6):567-86. doi: 10.1080/009841096161546. PMID: 8614024.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"orphanet-journal-of-rare-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ojrd","sideBox":"Learn more about [Orphanet Journal of Rare Diseases](http://ojrd.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ojrd/default.aspx","title":"Orphanet Journal of Rare Diseases","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"NGLY1, NGLY1 Deficiency, Rare diseases, Motor function, CNS, Mortality","lastPublishedDoi":"10.21203/rs.3.rs-7014298/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7014298/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eN-glycanase 1 (NGLY1) Deficiency is an ultra-rare autosomal recessive disorder of deglycosylation caused by loss-of-function mutations in the \u003cem\u003eNGLY1\u003c/em\u003e gene. Patient symptoms are characterized by developmental delay, intellectual disability, hyperkinetic movement disorder, elevated liver enzymes, (hypo)alacrima, and peripheral neuropathy. Despite supportive care, affected individuals often exhibit neurological deterioration at a young age, with caregivers reporting loss of previously attained motor skills by adolescence. Additionally, life-threatening complications are not uncommon, and the published median lifespan of patients is ~\u0026thinsp;15 years. The pathophysiology of NGLY1 Deficiency remains poorly understood, in part due to limited long-term studies in animal models. Notably, \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (C57BL/6) are embryonically lethal, and prior characterization of \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats was restricted to young adult rat (~\u0026thinsp;7 months old) before sacrifice, leaving any late-onset disease phenotypes or understanding of the potential for shortened lifespan unexamined.\u003c/p\u003e\u003cp\u003eIn the study reported here, longitudinal assessments of phenotypes in \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats were conducted alongside \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e controls. Survival, motor function, biochemical disease biomarkers, and histopathology of brain tissues were monitored in the rats from approximately 6 months to 17\u0026ndash;18 months of age.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e: \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats exhibited markedly reduced lifespan and progressive decline in both neurological behavior and quality of life compared with \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e rats. By 9\u0026ndash;10 months of age, ~\u0026thinsp;50% of the \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats had either died or met humane euthanasia criteria due to a severe decline in health. Surviving \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats showed other phenotypes mirroring human NGLY1 Deficiency disease progression, such as worsening motor deficits and wide-spread neuroinflammation. In contrast, heterozygous and wild-type littermates remained healthy and exhibited normal lifespan and aging profiles. Furthermore, histopathological examination of \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats identified significant neuropathological abnormalities not present in the control cohorts, including loss of peripheral axons and spinal motor neurons.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e: The findings reported here demonstrate that \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats recapitulate the severe, progressive course of NGLY1 Deficiency, including neurodegenerative deterioration, motor deficits, and premature mortality. This assessment of phenotypes and histology in \u003cem\u003eNgly1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e rats over an extended period of time provides valuable insights with respect to disease progression and lifespan in human patients.\u003c/p\u003e","manuscriptTitle":"Progressive Neurodegeneration, Motor Decline, and Premature Mortality in Aging Ngly1 Deficient Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-15 06:30:10","doi":"10.21203/rs.3.rs-7014298/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2025-10-14T16:34:38+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-09-08T02:25:17+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-08T01:22:26+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Orphanet Journal of Rare Diseases","date":"2025-07-04T06:30:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-04T03:02:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Orphanet Journal of Rare Diseases","date":"2025-07-02T15:58:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"orphanet-journal-of-rare-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ojrd","sideBox":"Learn more about [Orphanet Journal of Rare Diseases](http://ojrd.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ojrd/default.aspx","title":"Orphanet Journal of Rare Diseases","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f81fe49d-e9dc-4b58-b523-5e3ee2a43e19","owner":[],"postedDate":"September 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T16:01:03+00:00","versionOfRecord":{"articleIdentity":"rs-7014298","link":"https://doi.org/10.1186/s13023-026-04261-1","journal":{"identity":"orphanet-journal-of-rare-diseases","isVorOnly":false,"title":"Orphanet Journal of Rare Diseases"},"publishedOn":"2026-02-20 15:57:17","publishedOnDateReadable":"February 20th, 2026"},"versionCreatedAt":"2025-09-15 06:30:10","video":"","vorDoi":"10.1186/s13023-026-04261-1","vorDoiUrl":"https://doi.org/10.1186/s13023-026-04261-1","workflowStages":[]},"version":"v1","identity":"rs-7014298","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7014298","identity":"rs-7014298","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}