Lysosome Lipid Storage Disorder in NCTR-BALB/c Mice I. Description of the Disease and Genetics MANFORD D. MORRIS, PhD, From the Departments ofPediatrics and Biochemistry, University of CHIDAMBARAM BHUVANESWARAN, PhD, Arkansas for Medical Sciences, Little Rock, Arkansas, and the HELEN SHIO, MA, and STANLEY FOWLER, PhD Laboratory of Biochemical Cytology, The Rockefeller University, New York, New York We describe a strain of BALB/c mice, designated creased, while a-lipoprotein content is decreased. Se- NCTR-BALB/c, carrying a new genetic disorder char- rum total cholesterol remains normal. The serum activ- acterized by excessive tissue deposition of cholesterol ities of asparate aminotransferase, creatine phospho- and phospholipid. The mice exhibit progressive inco- kinase, and N-acetyl-p-glucosaminidase are elevated. ordination, grow less rapidly, and die 80-120 days after Free cholesterol levels are increased 8-10-fold in liver, birth. In comparison with control animals of the same spleen, and thymus, and about 2-fold in other tissues; age, organ weights in the affected animals are lower in but esterified cholesterol levels are normal. The phos- absolute value but higher relative to body weight, ex- pholipid content of several tissues is increased 50-100%, cept for the thymus, which is atrophied, and for the largely as a result of an increase in sphingomyelin con- lung and testes, whose absolute weights are not changed. tent. Significant increases in phosphatidylcholine occur Vacuolated cells are found in many tissues, and large also in spleen and lung. The disorder is inherited, af- foam cells are present in reticuloendothelial system fecting both sexes equaliy, and appears to be transmitted (RES)-rich organs. Compared with those of BALB/c as an autosomal recessive mutation. (AmJ Pathol 1982, controls, serum lipoproteins migrate more slowly on 108:140-149) electrophoresis; the amount of P-lipoproteins is in- WE HAVE DESCRIBED in preliminary articles"'2 a genetic defect, however, has not been established, strain of BALB/c mice that are affected with an in- and several unusual features of the mouse disorder herited disorder involving progressive neurologic hint at an etiology different from that of typical dysfunction, wasting, and premature death. Tissues Niemann-Pick disease. of the affected mice contained excessive amounts of unesterified cholesterol, and foam cells were seen in liver, spleen, thymus, lung, and gut. A colony of * The strain was derived from a colony of BALB/c mice at the National Center for Toxicological Research, Jeffer- these mice, which we refer to as the NCTR strain,* son, Arkansas. has now been produced, permitting a detailed study Supported by research grants AM-21376 and HL-18157 of the disease. In the papers presented here, we show from the National Institutes of Health and PCM-8008713 that these mice are affected by a generalized lysosome from the National Science Foundation. Dr. Fowler con- lipid storage disorder, and that unesterfied choles- ducted these studies during the tenure of an Otto G. Storm Established Investigatorship from the American Heart terol and sphingomyelin are principal accumulation Association. products. Activities of several lysosomal acid hydro- Accepted for publication March 10, 1982. lases were found increased in tissues of these mice, Dr. Fowler's present address is Department of Pathology, but acid sphingomyelinase activity was decreased. School of Medicine, University of South Carolina, Colum- These findings resemble many of those associated bia, SC 29208. Address reprint requests to Dr. Manford D. Morris, with Niemann-Pick disease in man,3 and they suggest Department of Pediatrics, University of Arkansas for that the NCTR-BALB/c mice could be an experi- Medical Sciences, 4301 W. Markham, Little Rock, Arkan- mental animal counterpart of the disease. The precise sas 72205. 0002-9440/82/0810-0140$01.30 © American Association of Pathologists 140 Vol. 108 * No. 2 LIPID STORAGE DISORDER IN NCTR-BALB/c MICE 141 The evidence supporting our conclusions is re- Animal Sacrifice and Autopsy ported in the accompanying articles,4'5 which The animals were lightly anesthetized with me- both studies on tissues of describe ultrastructural thoxyflurane (Metofane, Pitman-Moore Incorporated, affected NCTR-BALB/c mice and biochemical Washington Crossing, NJ). Blood was obtained from of storage inclusions isolated analyses purified by the heart after opening of the chest. After clotting, subcellular fractionation of livers from these mice. In serum was separated at 40 C, and 5,5'-dithiobis-(2- the present article we describe the main symptoms of nitrobenzoic acid) was added to a portion to inhibit the disorder, including clinical, gross, histopatho- logic, and biochemical findings. Evidence that the serum lecithin cholesterol acyltransferase.7 Organs and tissues were immediately excised, weighed, and disorder is transmitted as an autosomal recessive analyzed while fresh. genetic trait is also presented. While this work was in progress, Pentchev et al6 reported biochemical and enzymic studies on the same strain of mice, and a Histology comparison of their findings with ours is presented in the third article of this series.5 Specimens of tissues were fixed in neutral-buffered formalin and were processed by standard histologic procedures, or were fixed in 2.5 %o glutaraldehyde and Materials and Methods embedded in Epon 812. Thick Epon sections (1 ,) were cut and stained with 0.5% methylene blue, Colony Source 0.5% azure II (Allied Chemical, Morristown, NJ) in All mice described in this report were derived from 0.5%o sodium borate. Photographs were taken with a the strain BALB/cStCrlfC3HfNctr currently in use Zeiss universal photomicroscope on Kodak Pana- at the National Center for Toxicological Research tomic-X film. (NCTR), Jefferson, Arkansas. The propositus mouse (female), discovered in March 1975, was one of Analytic Procedures about 24,000 mice that were used in the 2-ami- noacetylfluorene effective dose 01 experiment con- Enzymes ducted at the NCTR. It was found moribund and ex- Serum aspartate aminotransferase8 and creatine hibited features suggesting a storage disorder (see phosphokinase9 were determined with a Rotochem II Results). The parents of the propositus mouse were a analyzer (Aminco Instruments, Silver Spring, Md). sibling breeding pair; another sibling breeding pair N-acetyl-p-glucosaminidase activity in serum was from the same litter also produced affected progeny. assayed by the method described in the accompany- The NCTR-BALB/c mouse colony described here ing article.5 was developed from these two breeding pairs, pri- marily by brother-sister matings, although early on, Lipids father-daughter matings were also carried out.t The Tissue Extraction: The weighed tissue was homog- control mice were derived from a separate colony ob- enized in a Polytron homogenizer (Brinkman In- tained from the BALB/c breeding stock at the struments, Westbury, NY), and was extracted three NCTR. times with a total of at least 50 volumes of chloro- form-methanol (2: 1). The pooled crude lipid ex- Colony Maintenance tracts were washed and purified by the method of Folch et al. Weanling mice were separated from their mothers Cholesterol: Tissue unesterified and esterified at 21-25 days of age, weighed, identified by toe clip, cholesterol and total serum cholesterol were de- and caged according to sex. Usually, 2-5 animals termined by the method of Rudel and Morris."' For were housed in a single plastic cage. They were allowed tissue total cholesterol, saponification was carried food (Purina Mouse Chow 5015Z) and water ad libi- out in 307o alcoholic KOH at 60 C for at least 30 tum. Affected NCTR-BALB/c mice were detected by minutes. Cholesterol was then extracted and quan- clinical signs and were retained with their littermates. titated by the procedure cited. For measurement of growth, the mice were weighed Phospholipid: Phospholipid phosphorus was de- daily to the nearest 0.1 g on an electronic balance. termined by the method of Fiske and Subbarow.'2 The phospholipid classes were separated by one- t Investigators wishing to acquire these mice should con- dimensional thin-layer chromatography on glass tact Dr. Manford D. Morris. plates coated with silica gel 60 F 254 (Merck and 142 MORRUS ET AL AJP * August 1982 Table 1 -Clinical Signs in Affected NCTR-BALB/c Mice* phosphatidylcholine, and a blank area were scraped Tremor of hind legs when walking and analyzed in every run. Jerky interrupted movements (staggering gait) Front feet spread apart when standing still Serum Electrophoresis Tendency to hold head higher than littermates Outward extension of forelimbs and rhythmic tremor Serum protein and lipoprotein electrophoresis in when held by the tail agarose was performed by a modification of the 13 Listed in order of usual appearance during growth and develop- method of Noble. Serum proteins were stained with ment of mice. 1% aqueous light green SF (Harleco, Philadelphia, Pa), and lipoproteins were stained at 400 C with 0.05% oil red 0 (Sigma Chemical Company) in 700o Company, Cincinnati, Ohio). The solvents used in ethanol. The serum samples were analyzed within 2 succession to separate the classes of phospholipids hours of bleeding. were 1) chloroform/methanol/water (16: 7: 1) and 2) chloroform/methanol/glacial acetic acid/water Results (11 :7.5:2:1).13 A standard mixture consisting of sphingomyelin, phosphatidylcholine, phosphatidyl- Signs and Symptoms of the Disorder inositol, phosphatidylethanolamine, and phospha- Table 1 lists the clinical symptoms exhibited by the tidylserine (Avanti Biochemicals, Birmingham, Ala) affected NCTR-BALB/c mice. The symptoms ap- was included in each run. After detection by iodine peared at variable periods during growth and, once followed by sublimation, the areas corresponding to present, developed progressively in intensity. Tremor the phospholipids were scraped and quantified."4 In of the back legs when walking was often the first symp- addition, the origin, an area corresponding to lyso- tom to appear, usually about 30-35 days after birth.