Mary Anna Thrall

Critical role for glycosphingolipids in Niemann-Pick disease type C

Niemann-Pick type C (NPC) disease is a cholesterol lipidosis caused by mutations in NPC1 and NPC2 gene loci. Most human cases are caused by defects in NPC1, as are the spontaneously occurring NPC diseases in mice and cats. NPC1 protein possesses a sterol-sensing domain and has been localized to vesicles that are believed to facilitate the recycling of unesterified cholesterol from late endosomes/lysosomes to the ER and Golgi. In addition to accumulating cholesterol, NPC1-deficient cells also accumulate gangliosides and other glycosphingolipids (GSLs), and neuropathological abnormalities in NPC disease closely resemble those seen in primary gangliosidoses. These findings led us to hypothesize that NPC1 may also function in GSL homeostasis. To evaluate this possibility, we treated murine and feline NPC models with N-butyldeoxynojirimycin (NB-DNJ), an inhibitor of glucosylceramide synthase, a pivotal enzyme in the early GSL synthetic pathway. Treated animals showed delayed onset of neurological dysfunction, increased average life span (in mice), and reduced ganglioside accumulation and accompanying neuropathological changes. These results are consistent with our hypothesis and with GSLs being centrally involved in the pathogenesis of NPC disease, and they suggest that drugs inhibiting GSL synthesis could have a similar ameliorating effect on the human disorder.

GABAergic neuroaxonal dystrophy and other cytopathological alterations in feline Niemann-Pick disease type C

Abstract Feline Niemann-Pick disease type C (NPC) is an autosomal recessive lysosomal storage disease which shares many of the clinical, biochemical and pathological features of the corresponding human disorder. Cytopathological alterations in distinct neuronal cell populations were investigated in this animal model to gain a better understanding of the pathogenesis of brain dysfunction. Golgi and immunocytochemical methods were employed to characterize the cell architectural changes occurring in neuronal somata, dendrites and axons at different stages of disease progression. Cortical pyramidal neurons in laminae II, III, and V exhibited various degrees of meganeurite and/or swollen axon hillock formation with or without ectopic dendritogenesis. Enlarged axon hillock regions with neuritic processes and spines were recognized early in the progression of feline NPC but were less prevalent in mid to late stages of the disease. Glutamic acid decarboxylase (GAD) immunocytochemistry demonstrated immunoreactive spheroids in numerous GABAergic axons in neocortex, subcortical areas, and cerebellum. Parvalbumin-immunoreactive axonal spheroid distribution in brain closely mirrored results from the GAD studies, whereas calbindin D-28k-immunoreactive spheroids were conspicuously absent in most cortical and subcortical areas examined. Purkinje cell axonal spheroid formation progressed in a distal to proximal direction, with eventual involvement of recurrent axon collaterals. Purkinje cell death and a concomitant decrease in the numbers of spheroids in the cerebellum were observed late in the disease course. Clinical neurological signs in feline NPC occur in parallel with neuronal structural alterations and suggest that GABAergic neuroaxonal dystrophy is a contributor to brain dysfunction in this disease.

Feline sphingolipidosis resembling Niemann-Pick disease type C

SummaryA 9-week old domestic short-hair kitten with progressive neurological dysfunction had histopathological lesions consistent with a lysosomal storage disease. Light microscopy of the brain, spinal cord, and ganglia revealed distention and vacuolation of many neuronal populations, and extensive neuroaxonal dystrophy. Large numbers of foamy macrophages were observed in the liver, spleen, lymph nodes, and lung. Hepatocytes appeared pale and swollen. Ultrastructural examination of all affected tissues and organs revealed heterogeneous membranous inclusions. Lipid analysis of liver revealed an excess of cholesterol, glucosylceramide, lactosylceramide and phospholipids including sphingomyelin. There was some increase in the levels of brain GM2 and GM3 gangliosides. Sphingomyelinase activity in liver was partially deficient or low normal. Skin fibroblasts were cultured from two affected cats from the colony established with littermates of the subject of this report. The cultured skin fibroblasts had partially decreased sphingomyelinase activity and a greatly decreased ability to esterify exogenous cholesterol. Clinical, morphological, and biochemical findings suggest that this cat had sphingolipidosis similar to human Niemann-Pick disease type C, a disease not previously described in the cat. The feline form of this storage disease may provide a useful model for studies on the human disease.

Mutation analysis of feline Niemann-Pick C1 disease.

Niemann-Pick C (NPC) disease is an autosomal recessive neurovisceral lysosomal storage disorder that results in defective intracellular transport of cholesterol. The major form of human NPC (NPC1) has been mapped to chromosome 18, the NPC1 gene (NPC1) has been sequenced and several mutations have been identified in NPC1 patients. A feline model of NPC has been characterized and is phenotypically, morphologically, and biochemically similar to human NPC1. Complementation studies using cultured fibroblasts from NPC affected cats and NPC1 affected humans support that the gene responsible for the NPC phenotype in this colony of cats is orthologous to human NPC1. Using human-based PCR primers, initial fragments of the feline NPC cDNA were amplified and sequenced. From these sequences, feline-specific PCR primers were generated and designed to amplify six overlapping bands that span the entire feline NPC1 open reading frame. A single base substitution (2864G-C) was identified in NPC1 affected cats. Obligate carriers are heterozygous at the same allele and a PCR-based assay was developed to identify the geneotype of all cats in the colony. The mutation results in an amino acid change from cysteine to serine (C955S). Several of the mutations identified in people occur in the same region. Marked similarity exists between the human and feline NPC1 cDNA sequences, and is greater than that between the human and murine NPC1 sequences. The human cDNA sequence predicts a 1278aa protein with a lysosomal targeting sequence, several trans-membrane domains and extensive homology with other known mediators of cholesterol homeostasis.

Comparison of the effects of ethanol and 4-methylpyrazole on the pharmacokinetics and toxicity of ethylene glycol in the dog☆

The purpose of this investigation was to compare the effects of ethanol and 4-methylpyrazole (4MP) on the toxicity and pharmacokinetics of ethylene glycol (EG) in the dog. All dogs received 173 mmol/kg EG, p.o. Dogs were randomly assigned to 3 groups: EG-treated only, EG + ethanol (19.3 mmol/kg, i.v. 3, 7, 14 and 24 h after EG) and EG + 4MP (0.24 mmol/kg, i.v. 3 h after EG, 0.18 mmol/kg at 24 h and 0.06 mmol/kg at 36 h). EG produced a rapid onset of metabolic acidosis (within 3 h) and acute oliguric renal failure (after 48 h), whereas administration of ethanol or 4MP greatly attenuated acidosis and prevented renal toxicity. The administration of ethanol, however, severely increased the central nervous system (CNS) depression that existed after ingestion of EG. The half-life of FG in serum was 10.8 +/- 0.7 h in the EG-only treatment group, 6.8 +/- 0.7 (P less than 0.05) in the EG + ethanol group and 9.8 +/- 0.9 h in the EG + 4MP group. Approx. 10% and 48% of the dose of EG was excreted unchanged in the urine at the 0-3 and 3-72 h periods, respectively. Treatment with 4MP increased the amount of EG excreted in the urine (71% from 3-72 h), whereas ethanol did not (51%). However, both ethanol and 4MP increased the rate constant of EG excretion into urine approx. 70%. These data demonstrate the utility of 4MP over ethanol for the treatment of EG-induced toxicity in dogs and indicate that ethanol and 4MP cause an increase in the rate constant of EG excretion in the urine and not a prolongation in EG half-life.

Abnormal neuronal metabolism and storage in mucopolysaccharidosis type VI (Maroteaux–Lamy) disease

Mucopolysaccharidosis (MPS) type VI, also known as Maroteaux–Lamy disease, is an inherited disorder of glycosaminoglycan  catabolism  caused  by  deficient  activity of the lysosomal hydrolase, N‐acetylgalactosamine 4‐sulphatase (4S). A variety of prominent visceral and skeletal defects are characteristic, but primary neurological involvement has generally been considered absent. We report here that the feline model of MPS VI exhibits abnormal lysosomal storage in occasional neurones and glia distributed throughout the cerebral cortex. Abnormal lysosomal inclusions were pleiomorphic with some resembling zebra bodies and dense core inclusions typical of other MPS diseases or the membranous storage bodies characteristic of the gangliosidoses. Pyramidal neurones were shown to contain abnormal amounts of GM2 and GM3 gangliosides by immunocytochemical staining and unesterified cholesterol by histochemical (filipin) staining. Further, Golgi staining of pyramidal neurones revealed that some possessed ectopic axon hillock neurites and meganeurites similar to those described in Tay–Sachs and other neuronal storage diseases with ganglioside storage. Some animals evaluated in this study also received allogeneic bone marrow transplants, but no significant differences in neuronal storage were noted between treated and untreated individuals. These studies demonstrate that deficiency of 4S activity can lead to metabolic abnormalities in the neurones of central nervous system in cats, and that these changes may not be readily amenable to correction by bone marrow transplantation. Given the close pathological and biochemical similarities between feline and human MPS VI, it is conceivable that children with this disease have similar neuronal involvement.

Effects of dietary cholesterol restriction in a feline model of Niemann–Pick type C disease

A feline model of Niemann–Pick disease type C (NPC) was employed to evaluate the effect of dietary cholesterol restriction on progression of disease. Two NPC-affected treated cats were fed a cholesterol-restricted diet beginning at 8 weeks of age; the cats remained on the diet for 150 and 270 days respectively. The study goal was to lower the amount of low density lipoprotein (LDL) available to cells, hypothetically reducing subsequent lysosomal accumulation of unesterified cholesterol and other lipids. Neurological progression of disease was not altered and dietary cholesterol restriction did not significantly decrease storage in NPC-affected treated cats. One NPC-affected treated cat had decreased serum alkaline phosphatase activity (ALP) and decreased serum cholesterol concentration. Liver lipid concentrations of unesterified cholesterol, cholesterol ester and phospholipids in NPC-affected treated cats were similar to those seen in NPC-affected untreated cats. Ganglioside concentrations in the NPC-affected treated cats and NPC-affected untreated cats were similar. Histological findings in liver sections from NPC-affected treated cats showed a diffuse uniform microvacuolar pattern within hepatocytes and Kupffer cells, in contrast to a heterogeneous macro/microvacuolar pattern and prominent nodular fibrosis in NPC-affected untreated cats. Similar differences in vacuolar patterns were seen in splenic macrophages. Although some hepatic parameters were modified, dietary cholesterol restriction did not appear to alter disease progression in NPC-affected kittens.

Metabolic abnormalities in feline Niemann-Pick type C heterozygotes

SummaryNiemann-Pick disease type C (NPC) is an autosomal recessive neurovisceral lysosomal storage disorder in which cholesterol lipidosis results from defective intracellular transport of unesterified cholesterol. The primary molecular defect of NPC is unknown; regulatory mechanisms of cholesterol metabolism are impaired, resulting in retarded esterification of exogenous cholesterol with accumulation of unesterified cholesterol in lysosomes and secondary storage of glycolipids and sphingomyelin. In obligate heterozygotes from a feline NPC model, cultured skin fibroblasts challenged with exogenously derived cholesterol exhibited intermediate rates of cholesterol esterification and accumulation of unesterified cholesterol. Liver lipid analyses of obligate heterozygote cats demonstrated intermediate cholesterol and sphingomyelin concentrations. Vacuolated skin fibroblasts were found in 2 of 3 heterozygote cats, and occasional cortical neurons exhibited intracellular inclusions immunoreactive for GM2-ganglioside. Ultrastructural studies provided evidence of storage in liver and brain. We believe these morphological and biochemical findings are the first example of manifestations of CNS abnormalities in a genetic carrier for a neuronal storage disease.

Safety and efficacy of high-dose fomepizole compared with ethanol as therapy for ethylene glycol intoxication in cats.

OBJECTIVE To determine the safety and efficacy of high-dose fomepizole compared with ethanol (EtOH) in cats with ethylene glycol (EG) toxicosis. DESIGN Prospective study. SETTING University veterinary research laboratory. ANIMALS Thirteen cats. INTERVENTIONS Two cats received injections of high-dose fomepizole (Study 1). Three cats received lethal doses of EG and fomepizole treatment was initiated 1, 2, or 3 hours later (Study 2). Eight cats received a lethal dose of EG and were treated with fomepizole or EtOH (Study 3). Cats treated with fomepizole received 125 mg/kg IV initially, then 31.25 mg/kg at 12, 24, and 36 hours. Cats treated with EtOH received 5 mL of 20% EtOH/kg IV initially, then every 6 hours for 5 treatments, then every 8 hours for 4 treatments. Cats also received fluids and supportive therapy as needed. MEASUREMENTS AND MAIN RESULTS Clinical signs were monitored and serial blood analyses performed. Cats receiving fomepizole experienced mild sedation but no biochemical evidence of toxicity. Cats receiving fomepizole for EG intoxication survived if therapy was initiated within 3 hours of EG ingestion. One of the 6 developed acute renal failure (ARF) but survived. Only 1 of the 3 cats treated with EtOH 3 hours following EG ingestion survived; 2 developed ARF and were euthanized. Cats treated 4 hours following EG ingestion developed ARF, whether treated with EtOH or fomepizole. CONCLUSIONS Fomepizole is safe when administered to cats in high doses, prevents EG-induced fatal ARF when therapy is instituted within 3 hours of EG ingestion, and is more effective than treatment with EtOH.

Urine glycosaminoglycan concentrations in mucopolysaccharidosis VI-affected cats following bone marrow transplantation or leukocyte infusion

Urinary glycosaminoglycan (GAG) concentrations were determined in nineteen normal cats (eleven kittens and eight adult cats), eighteen mucopolysaccharidosis VI (MPS VI)-affected untreated cats (ten kittens and eight adult cats), thirteen cats MPS VI-affected cats following bone marrow transplants (BMT), and two MPS VI-affected cats following intravenous infusion of leukocytes from normal cats. Mucopolysaccharidosis VI-affected cats treated with BMT had a precipitous decrease in urinary GAG by day 7 post-BMT, then a transient increase just prior to engraftment, followed by a sustained decrease to within, or near, the range of urinary GAG concentration established for normal cats. The pre-engraftment changes in urinary GAG excretion were reproduced by leukocyte infusion. After infusion of comparable members of normal peripheral blood leukocytes, a significant decrease in urinary GAG concentrations, specifically dermatan sulfate (DS), was seen with a nadir at day 5 post-infusion, followed by a return by day 9 to pre-infusion values. Post-engraftment, a continued low urinary GAG concentration with a specific decrease in DS can be utilized to document successful autologous engraftment in MPS VI-affected cats.