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

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

Mutation in Smek2 regulating hepatic glucose metabolism causes hypersarcosinemia and hyperhomocysteinemia in rats.

Suppressor of mek1 (Dictyostelium) homolog 2 (Smek2), was identified as one of the responsible genes for diet-induced hypercholesterolemia (DIHC) of exogenously hypercholesterolemic (ExHC) rats. A deletion mutation in Smek2 leads to DIHC via impaired glycolysis in the livers of ExHC rats. The intracellular role of Smek2 remains obscure. We used microarrays to investigate Smek2 functions with ExHC and ExHC.BN-Dihc2BN congenic rats that harbor a non-pathological Smek2 allele from Brown-Norway rats on an ExHC background. Microarray analysis revealed that Smek2 dysfunction leads to extremely low sarcosine dehydrogenase (Sardh) expression in the liver of ExHC rats. Sarcosine dehydrogenase demethylates sarcosine, a byproduct of homocysteine metabolism. The ExHC rats with dysfunctional Sardh developed hypersarcosinemia and homocysteinemia, a risk factor for atherosclerosis, with or without dietary cholesterol. The mRNA expression of Bhmt, a homocysteine metabolic enzyme and the hepatic content of betaine (trimethylglycine), a methyl donor for homocysteine methylation were low in ExHC rats. Results suggest that homocysteine metabolism rendered fragile by a shortage of betaine results in homocysteinemia, and that Smek2 dysfunction causes abnormalities in sarcosine and homocysteine metabolism.

Physiological importance of NGLY1, as revealed by rodent model analyses.

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

Loss of peptide:N-glycanase causes proteasome dysfunction mediated by a sugar-recognizing ubiquitin ligase.

Mutations in the human peptide:N-glycanase gene (NGLY1), which encodes a cytosolic de-N-glycosylating enzyme, cause a congenital autosomal recessive disorder. In rodents, the loss of Ngly1 results in severe developmental delay or lethality, but the underlying mechanism remains unknown. In this study, we found that deletion of Fbxo6 (also known as Fbs2), which encodes a ubiquitin ligase subunit that recognizes glycoproteins, rescued the lethality-related defects in Ngly1-KO mice. In NGLY1-KO cells, FBS2 overexpression resulted in the substantial inhibition of proteasome activity, causing cytotoxicity. Nuclear factor, erythroid 2-like 1 (NFE2L1, also known as NRF1), an endoplasmic reticulum-associated transcriptional factor involved in expression of proteasome subunits, was also abnormally ubiquitinated by SCFFBS2 in NGLY1-KO cells, resulting in its retention in the cytosol. However, the cytotoxicity caused by FBS2 was restored by the overexpression of "glycan-less" NRF1 mutants, regardless of their transcriptional activity, or by the deletion of NRF1 in NGLY1-KO cells. We conclude that the proteasome dysfunction caused by the accumulation of N-glycoproteins, primarily NRF1, ubiquitinated by SCFFBS2 accounts for the pathogenesis resulting from NGLY1 deficiency.

Reversibility of motor dysfunction in the rat model of NGLY1 deficiency.

N-glycanase 1 (NGLY1) deficiency is a rare inherited disorder characterized by developmental delay, hypolacrima or alacrima, seizure, intellectual disability, motor deficits, and other neurological symptoms. The underlying mechanisms of the NGLY1 phenotype are poorly understood, and no effective therapy is currently available. Similar to human patients, the rat model of NGLY1 deficiency, Ngly1-/-, shows developmental delay, movement disorder, somatosensory impairment, scoliosis, and learning disability. Here we show that single intracerebroventricular administration of AAV9 expressing human NGLY1 cDNA (AAV9-hNGLY1) to Ngly1-/- rats during the weaning period restored NGLY1 expression in the brain and spinal cord, concomitant with increased enzymatic activity of NGLY1 in the brain. hNGLY1 protein expressed by AAV9 was found predominantly in mature neurons, but not in glial cells, of Ngly1-/- rats. Strikingly, intracerebroventricular administration of AAV9-hNGLY1 normalized the motor phenotypes of Ngly1-/- rats assessed by the rota-rod test and gait analysis. The reversibility of motor deficits in Ngly1-/- rats by central nervous system (CNS)-restricted gene delivery suggests that the CNS is the primary therapeutic target organs for NGLY1 deficiency, and that the Ngly1-/- rat model may be useful for evaluating therapeutic treatments in pre-clinical studies.

JF1/B6F1 Ngly1-/- mouse as an isogenic animal model of NGLY1 deficiency.

N-Glycanase 1 (NGLY1) deficiency is a congenital disorder caused by mutations in the NGLY1 gene. Because systemic Ngly1-/- mice with a C57BL/6 (B6) background are embryonically lethal, studies on the mechanism of NGLY1 deficiency using mice have been problematic. In this study, B6-Ngly1-/+ mice were crossed with Japanese wild mice-originated Japanese fancy mouse 1 (JF1) mice to produce viable F2 Ngly1-/- mice from (JF1×B6)F1 Ngly1-/+ mice. Systemic Ngly1-/- mice with a JF1 mouse background were also embryonically lethal. Hybrid F1 Ngly1-/- (JF1/B6F1) mice, however, showed developmental delay and motor dysfunction, similar to that in human patients. JF1/B6F1 Ngly1-/- mice showed increased levels of plasma and urinary aspartylglycosamine, a potential biomarker for NGLY1 deficiency. JF1/B6F1 Ngly1-/- mice are a useful isogenic animal model for the preclinical testing of therapeutic options and understanding the precise pathogenic mechanisms responsible for NGLY1 deficiency.

Ngly1 -/- rats develop neurodegenerative phenotypes and pathological abnormalities in their peripheral and central nervous systems.

N-glycanase 1 (NGLY1) deficiency, an autosomal recessive disease caused by mutations in the NGLY1 gene, is characterized by developmental delay, hypolacrima or alacrima, seizure, intellectual disability, movement disorders and other neurological phenotypes. Because of few animal models that recapitulate these clinical signatures, the mechanisms of the onset of the disease and its progression are poorly understood, and the development of therapies is hindered. In this study, we generated the systemic Ngly1-deficient rodent model, Ngly1-/- rats, which showed developmental delay, movement disorder, somatosensory impairment and scoliosis. These phenotypes in Ngly1-/- rats are consistent with symptoms in human patients. In accordance with the pivotal role played by NGLY1 in endoplasmic reticulum-associated degradation processes, cleaving N-glycans from misfolded glycoproteins in the cytosol before they can be degraded by the proteasome, loss of Ngly1 led to accumulation of cytoplasmic ubiquitinated proteins, a marker of misfolded proteins in the neurons of the central nervous system of Ngly1-/- rats. Histological analysis identified prominent pathological abnormalities, including necrotic lesions, mineralization, intra- and extracellular eosinophilic bodies, astrogliosis, microgliosis and significant loss of mature neurons in the thalamic lateral and the medial parts of the ventral posterior nucleus and ventral lateral nucleus of Ngly1-/- rats. Axonal degradation in the sciatic nerves was also observed, as in human subjects. Ngly1-/- rats, which mimic the symptoms of human patients, will be a useful animal model for preclinical testing of therapeutic options and understanding the detailed mechanisms of NGLY1 deficiency.

Low utilization of glucose in the liver causes diet-induced hypercholesterolemia in exogenously hypercholesterolemic rats.

Exogenously hypercholesterolemic (ExHC) rats develop diet-induced hypercholesterolemia (DIHC) when fed with dietary cholesterol. Previously, we reported that, under the high-sucrose-diet-feeding condition, a loss-of-function mutation in Smek2 results in low activity of fatty acid synthase (FAS) followed by the shortage of hepatic triacylglycerol content in ExHC rats and the onset of DIHC. However, the relationship between the Smek2 mutation and FAS dysfunction is still unclear. Here, we focused on carbohydrate metabolism, which provides substrates for FAS, and analyzed carbohydrate and lipid metabolisms in ExHC rats to clarify how the deficit of Smek2 causes DIHC. Male ExHC and SD rats were fed high-sucrose or high-starch diets containing 1% cholesterol for 2 weeks. Serum cholesterol levels of the ExHC rats were higher, regardless of the dietary carbohydrate. Hepatic triacylglycerol levels were higher in only the SD rats fed the high-sucrose diet. Moreover, the ExHC rats exhibited a diabetes-like status and accumulation of hepatic glycogen and low hepatic mRNA levels of liver-type phosphofructokinase (Pfkl), which encodes a rate-limiting enzyme for glycolysis. These results suggest that the glucose utilization, particularly glycolysis, is impaired in the liver of ExHC rats. To evaluate how the diet with extremely low glucose affect to DIHC, ExHC.BN-Dihc2BN, a congenic strain that does not develop DIHC, and ExHC rats were fed a high-fructose diet containing 1% cholesterol for 2 weeks. The serum cholesterol and hepatic triacylglycerol levels were similar in the strains. Results of water-soluble metabolite analysis with primary hepatocytes, an increase in fructose-6-phosphate and decreases in succinate, malate and aspartate in ExHC rats, support impaired glycolysis in the ExHC rats. Thus, the Smek2 mutation causes abnormal hepatic glucose utilization via downregulation of Pfkl expression. This abnormal glucose metabolism disrupts hepatic fatty acid synthesis and causes DIHC in the ExHC rats.

Establishment of X-linked Alport syndrome model mice with a Col4a5 R471X mutation.

Alport syndrome (AS) is an inherited disorder characterized by glomerular basement membrane (GBM) abnormality and development of chronic kidney disease at an early age. The cause of AS is a genetic mutation in type IV collagen, and more than 80% of patients have X-linked AS (XLAS) with mutation in COL4A5. Although the causal gene has been identified, mechanisms of progression have not been elucidated, and no effective treatment has been developed. In this study, we generated a Col4a5 mutant mouse harboring a nonsense mutation (R471X) obtained from a patient with XLAS using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated system. Col4a5 mRNA and protein expressions were not observed in the kidneys of hemizygous R471X male mice. R471X mice showed proteinuria and hematuria. Pathology revealed progression of glomerulosclerosis and interstitial fibrosis by age. Electron microscopy identified irregular thickening in GBM accompanied by irregular lamination. These observations were consistent with the clinical and pathological features of patients with AS and other established models. In addition, our mice models develop end-stage renal disease at the median age of 28 weeks, much later compared to previous models much more consistent with clinical course of human XLAS. Our models have advantages for future experiments in regard with treatment for human XLAS.

Osborne-Mendel rats simultaneously develop cardiac and renal dysfunction, left atrial thrombosis, peripheral artery occlusion, and ascending aortic dissection.

Although chronic kidney disease (CKD) is strongly associated with onsets of cardiovascular disease (CVD), the pathogenic mechanism between these diseases has not been fully understood. To develop and validate new therapeutic strategies for this complication, appropriate experimental models that reflect the complexity of the underlying pathophysiology are needed. The Osborne-Mendel (OM) rat was identified as an atherosclerosis-prone and a premature-death rat strain among 16 inbred rat strains when fed high-cholesterol containing diet. When fed high-cholesterol diet, OM rats showed simultaneous occurrence of aortic aneurysm, aortic dissection, peripheral artery occlusion, and left atrial thrombosis. OM rats had significantly lower max dP/dt and higher min dP/dt than F344 rats did, indicating impaired left ventricle contractility and relaxation. OM rats developed renal dysfunction, showing increased urinary albumin excretion. OM rats also showed mild hypertension, decreased endothelial function, and enhanced coagulation and platelet aggregation, compared with F344 rats. We now report that OM rat would be a novel spontaneous animal model which simultaneously demonstrates cardiac and renal dysfunction, and CVD events. This model could be a useful model for the pre-clinical testing of pharmacological therapies and could provide new insight into potential targets and pathways for the treatment of CKD and CVD.

Human apolipoprotein B transgenic SHR/NDmcr-cp rats show exacerbated kidney dysfunction.

Nephropathy frequently co-occurs with metabolic syndrome in humans. Metabolic syndrome is a cluster of metabolic diseases including obesity, diabetes, hypertension, and dyslipidemia, and some previous studies revealed that dyslipidemia contributes to the progression of kidney dysfunction. To establish a new nephropathy model with metabolic syndrome, we produced human apolipoprotein B (apoB) transgenic (Tg.) SHR/NDmcr-cp (SHR-cp/cp) rats, in which dyslipidemia is exacerbated more than in an established metabolic syndrome model, SHR-cp/cp rats. Human apoB Tg. SHR-cp/cp rats showed obesity, hyperinsulinemia, hypertension, and severe hyperlipidemia. They also exhibited exacerbated early-onset proteinuria, accompanied by increased kidney injury and increased oxidative and inflammatory markers. Histological analyses revealed the characteristic features of human apoB Tg. SHR-cp/cp rats including prominent glomerulosclerosis with lipid accumulation. Our newly established human apoB Tg. SHR-cp/cp rat could be a useful model for the nephropathy in metabolic syndrome and for understanding the interaction between dyslipidemia and renal dysfunction in metabolic syndrome.

Unavailability of liver triacylglycerol increases serum cholesterol concentration induced by dietary cholesterol in exogenously hypercholesterolemic (ExHC) rats.

BACKGROUND: Exogenously hypercholesterolemic (ExHC) rats develop hypercholesterolemia and low hepatic triacylglycerol (TAG) levels when dietary cholesterol is loaded. The responsible gene Smek2 was identified via linkage analysis using the original strain Sprague-Dawley (SD) rats. In this study, we compared SD and ExHC rats to investigate a relationship between hypercholesterolemia and the low hepatic TAG levels observed in ExHC rats. METHODS: Male 4-weeks-old ExHC and SD rats were fed a 1% cholesterol diet for 1 week. Serum and liver parameters were analyzed. Gene expression and enzyme activities related to TAG metabolism were also assessed. RESULTS: We reproducibly observed higher serum cholesterol and lower hepatic TAG levels in ExHC rats than in SD rats. Golgi apparatus in the livers of ExHC rats secreted ß-very-low-density lipoprotein (ß-VLDL) that had higher cholesterol ester (CE) and lower TAG content than those in the ß-VLDL secreted by SD rats. Gene expression related to fatty acid and TAG synthesis in ExHC rats was lower than that in SD rats. Enzymatic activities for fatty acid synthesis were also relatively lower in ExHC rats. Moreover, the fatty acid composition of hepatic and serum CE in ExHC rats showed that these CEs were not modified after secretion from the liver despite the similar activities of serum lecithin-cholesterol acyltransferase (LCAT) in ExHC rats to those in SD rats. CONCLUSIONS: Low production of liver TAG and secretion of CE-rich, TAG-poor ß-VLDL without modification by LCAT in the circulation contributed to hypercholesterolemia induced by dietary cholesterol in ExHC rats.

Hypercholesterolemia and atherosclerosis in low density lipoprotein receptor mutant rats.

To establish low density lipoprotein receptor (LDLR) mutant rats as a hypercholesterolemia and atherosclerosis model, we screened the rat LDLR gene for mutations using an N-ethyl-N-nitrosourea mutagenesis archive of rat gene data, and identified five mutations in its introns and one missense mutation (478T>A) in exon 4. The C160S mutation was located in the ligand binding domain of LDLR and was revealed to be equivalent to mutations (C160Y/G) identified in human familial hypercholesterolemia (FH) patients. The wild type, heterozygous, and homozygous mutant rats were fed a normal chow diet or a high fat high cholesterol (HFHC) diet from the age of 10 weeks for 16 weeks. The LDLR homozygous mutants fed the normal chow diet showed higher levels of plasma total cholesterol and LDL cholesterol than the wild type rats. When fed the HFHC diet, the homozygous mutant rats exhibited severe hyperlipidemia and significant lipid deposition from the aortic arch to the abdominal aorta as well as in the aortic valves. Furthermore, the female homozygous mutants also developed xanthomatosis in their paws. In conclusion, we suggest that LDLR mutant rats are a useful novel animal model of hypercholesterolemia and atherosclerosis.

Identification of SMEK2 as a candidate gene for regulation of responsiveness to dietary cholesterol in rats.

We have previously mapped a diet-induced hypercholesterolemia locus (Dihc2) to chromosome 14 in the F2 generation cross of high-responsive exogenous hypercholesterolemia rats and low-responsive BN rats. To identify a causal gene within this locus, we constructed interval-specific congenic lines and carried out expression and sequencing analyses. Here we narrowed Dihc2 to a region including 33 genes and predicted transcripts and identified RGD1309450_predicted, a homologous gene of SMEK2, as a strong candidate for responsiveness to dietary cholesterol. Our finding provides new insights into the pathway underlying the individual responsiveness to dietary cholesterol in vivo.

Genetic analysis of diet-induced hypercholesterolemia in exogenously hypercholesterolemic rats.

The exogenously hypercholesterolemic (ExHC) rat is an established strain that exhibits a polygenic syndrome of hypercholesterolemia after feeding on a cholesterol-containing diet, and the extent of this differs between male and female rats in the strain. The present study was performed to determine the genetic background of diet-induced hypercholesterolemia in ExHC rats. We used quantitative trait locus (QTL) analyses of the F2 progeny derived from ExHC and Brown-Norway rats. Rats were fed a diet containing 1% cholesterol, and a genome-wide scan was then performed. Significant QTLs for serum total cholesterol levels were revealed on chromosomes 5 and 14 in the vicinity of markers D5Rat95 and D14Rat43, having maximum logarithm of the odds scores of 6.0 and 5.8, respectively. A suggestive QTL for the trait was also detected on chromosome 3 at D3Rat140. In particular, the QTL on chromosome 5 was specific for female rats. These loci were novel QTLs for post-dietary serum total cholesterol levels. In addition, cross-mating analysis in F1 generations suggested that the responsiveness to dietary cholesterol in ExHC rats is partly attributable to X-linked inheritance. Identifying such genetic factors may be useful in predicting the risks associated with diet-induced hypercholesterolemia in humans.