Biochemical and Biophysical Research Communications 413 (2011) 400–406

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Biochemical and Biophysical Research Communications

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Npc1 deficiency in the C57BL/6J genetic background enhances Niemann–Pickdisease type C spleen pathology

Julio Parra 1, Andrés D. Klein 1, Juan Castro, María Gabriela Morales, Matías Mosqueira, Ilse Valencia,Victor Cortés, Attilio Rigotti, Silvana Zanlungo ⇑Departamento de Gastroenterología, Facultad de Medicina, Pontificia Universidad Católica, Santiago, Chile

a r t i c l e i n f o a b s t r a c t

Article history:Received 17 August 2011Available online 2 September 2011

Keywords:Niemann–Pick type C diseaseNPC1CholesterolLiverSpleen

0006-291X/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.bbrc.2011.08.096

Abbreviations: NPC, Niemann–Pick type C; NPC1,⇑ Corresponding author. Address: Departamento de

Universidad Católica de Chile, Marcoleta 367, Casilla+56 2 639 7780.

E-mail address: silvana@med.puc.cl (S. Zanlungo).1 These authors contributed equally.

Niemann–Pick type C (NPC) disease is an autosomal recessive neurovisceral lipid storage disorder. Theaffected genes are NPC1 and NPC2. Mutations in either gene lead to intracellular cholesterol accumula-tion. There are three forms of the disease, which are categorized based on the onset and severity ofthe disease: the infantile form, in which the liver and spleen are severely affected, the juvenile form,in which the liver and brain are affected, and the adult form, which affects the brain. In mice, a sponta-neous mutation in the Npc1 gene originated in the BALB/c inbred strain mimics the juvenile form of thedisease. To study the influence of genetic background on the expression of NPC disease in mice, we trans-ferred the Npc1 mutation from the BALB/c to C57BL/6J inbred background. We found that C57BL/6J-Npc1�/� mice present with a much more aggressive form of the disease, including a shorter lifespan thanBALB/c-Npc1�/� mice. Surprisingly, there was no difference in the amount of cholesterol in the brains ofNpc1�/� mice of either mouse strain. However, Npc1�/� mice with the C57BL/6J genetic backgroundshowed striking spleen damage with a marked buildup of cholesterol and phospholipids at an earlyage, which correlated with large foamy cell clusters. In addition, C57BL/6J Npc1�/�mice presented red cellabnormalities and abundant ghost erythrocytes that correlated with a lower hemoglobin concentration.We also found abnormalities in white cells, such as cytoplasmic granulation and neutrophil hyperseg-mentation that included lymphopenia and atypias. In conclusion, Npc1 deficiency in the C57BL6/J back-ground is associated with spleen, erythrocyte, and immune system abnormalities that lead to a reducedlifespan.

� 2011 Elsevier Inc. All rights reserved.

1. Introduction

Niemann–Pick type C (NPC) disease is a fatal autosomal reces-sive lipid storage disorder [1,2]. NPC1 and NPC2 are the affectedgenes [3,4]. NPC1 encodes a late endosomal transmembrane pro-tein that binds cholesterol in a luminal domain, and NPC2 encodesa small soluble lysosomal cholesterol binding and transport pro-tein [3–6]. Mutations in either of the NPC proteins cause intracel-lular accumulation of cholesterol and glycosphingolipids in lateendosomes and lysosomes in all tissues [7,8].

The initial clinical manifestation of NPC is neonatal jaundiceand an enlarged liver and/or spleen, but the disease manifestationsare very heterogeneous with respect to time of appearance and

ll rights reserved.

Niemann–Pick C1 protein.Gastroenterología, Pontificia114-D, Santiago, Chile. Fax:

progression [1,9,10]. Three different forms of the disease can bedistinguished: the infantile form, in which hepatosplenomegalyis almost invariably present and patients die before the age of 5;the classic disease with neurological onset during childhood andadolescence, in which neurodegeneration and motor coordinationproblems are hallmarks of the disease and majority of patientsdie in their teenage years; and the adult form, in which late neuro-logic symptomatology and progressive dementia are characteristic,and patients live into adulthood [1,9,10].

Mutations that inactivate NPC1 have been described not onlyin humans but also in other species, including mice [11,12]. Twomurine models of NPC1 disease, BALB/c npcnih and C57BL/KsJ spm(sphingomyelinosis or spm), arose by spontaneous mutation[11–13]. Both of the murine NPC1 models were found to be allelicby crossbreeding and belong to the same complementation groupas human NPC1 [14].

The Npc1 mutation in the BALB/c npcnih murine model results inearly protein truncation [15]. This model has been extensively uti-lized and characterized. In this model, animals present with highlevels of neuron loss, particularly of Purkinje cells in the

J. Parra et al. / Biochemical and Biophysical Research Communications 413 (2011) 400–406 401

cerebellum, and die between 70 and 85 days of age [15–18]. Thepathological changes observed in this model resemble those ob-served in the classical juvenile form of NPC disease.

This study was undertaken to analyze the relevance of geneticbackground in the expression of the most-studied Npc1 mutationin mice. To accomplish this, we transferred the Npc1-npcnih muta-tion from BALB/c into the C57BL/6J genetic background. We foundthat C57BL/6J-Npc1�/� mice have a shorter lifespan and veryaggressive spleen pathology that correlates with red and white cellabnormalities. In conclusion, C57BL/6J-Npc1�/� mice show a signif-icantly more aggressive form of NPC disease.

2. Materials and methods

2.1. Animals and diet

BALB/c mice carrying a heterozygous mutation in the NPC1gene [15] were kindly donated by Dr. Peter Pentchev from theNIH (Bethesda, MD) and were used to generate control (Npc1+/+)and homozygous mutant (Npc1�/�) animals. The Npc1 mutationwas transferred from the BALB/c background to C57BL/6J by suc-cessive backcrossing of Npc1+/� offspring with C57BL/6J mice ob-tained from the Jackson Laboratory (Bar Harbor, ME) for ninegenerations. Genotypes were identified using PCR-based screening,as previously described [15,19]. Mice had free access to water anda chow diet (<0.02% cholesterol, Prolab RMH 3000, PMI Feeds Inc.,St. Louis, MO). All protocols were approved by our institution’s re-view board for animal studies and were in agreement with US Pub-lic Health Service Policy on the Humane Care and Use of LaboratoryAnimals recommended by the Institute for Laboratory Animal Re-search in its ‘‘Guide for the Care and Use of Laboratory Animals’’.

2.2. Brain, liver, and spleen histology

Brain, liver and spleen tissues were fixed in 4% paraformalde-hyde for 48 h and then embedded in paraffin, sectioned, and placedon glass slides. Hematoxylin and eosin staining was then per-formed according to standard procedures.

2.3. Calbindin and filipin analysis

Four-week-old animals were sacrificed and perfused intracardi-ally with 0.9% NaCl followed by 4% paraformaldehyde in PBS. Fixedbrains were cut into 40-lm coronal sections in a cryostat at �20 �C(Leitz 1900). Cerebellum was analyzed by immunohistochemistrywith the anti-calbindin antibody D-28K (1:1000) (Chemicon Inter-national, Temecula, CA) by histological staining on floating sec-tions. Immunocytochemistry was performed using the avidinbiotin horseradish peroxidase complex (ABC) method (Vector Lab-oratories, Burlingame, CA), and sections were mounted on gelatinpre-coated slides, air-dried and, finally, covered with Entellan(Merck, Darmstadt, Germany).

Filipin staining was performed as previously described [18]. Thesections were analyzed using a fluorescent microscope (OlympusBX51).

2.4. Blood sampling and cell and hemoglobin analysis

Mice were anesthetized by intraperitoneal injection of keta-mine and xylazine at 80–100 and 5–10 mg/kg, respectively. Bloodwas obtained as previously described [20]. Peripheral blood smearswere made by placing a drop of blood on one end of a slide andusing a spreader slide to disperse the blood over the slide’s length.The slide was left to air dry after which the blood was fixed andstained with hematoxylin and eosin according to standard proce-

dures. The slides were then observed with a conventional micro-scope in the region where the cells were spaced far enough apartto be counted and differentiated. Blood cell counts and hemoglobinlevels were measured with an automatic counter.

2.5. Lipid analyses

Brain, liver and spleen tissues were obtained and stored at�80 �C. Cholesterol and phospholipid levels were determinedusing routine methods [21,22].

2.6. Statistics

The statistical significance of the difference between the meansof the experimental groups was evaluated using one-way analysisof variance (ANOVA) with a post hoc Tukey multiple-comparisontest (Prism 3.0, GraphPad). A difference was considered statisticallysignificant at a p value < 0.05.

3. Results

3.1. Lifespan and growth curves for BALB/c- and C57BL/6J-Npc1�/�

mice

To study the role of genetic background in the progression of NPCdisease, we transferred the Npc1nih mutation from the original BALB/c mouse strain to the C57BL/6J strain by successive backcrossing ofthe offspring. We first analyzed lifespan. Survival curves revealed adramatic difference between the genetic backgrounds. WhileBALB/c-Npc1�/� mice died between 70 and 85 days, C57BL/6J-Npc1�/� mice died between 28 and 35 days (Fig. 1A). Next, wemeasured mouse weight. As has been previously described, BALB/c-Npc1�/�mice gain weight until they are 6–7 weeks old, after whichthey begin to decline in weight (Fig. 1B) [18,23]. C57BL/6J-Npc1�/�

mice were significantly smaller than BALB/c-Npc1�/� mice at allages. In addition, C57BL/6J-Npc1�/� mice start to lose weight whenthey are 2–3 weeks old (Fig. 1B). These results indicate that geneticbackground has a strong influence on the NPC phenotype.

3.2. Organ weights and lipid content of BALB/c- and C57BL/6J-Npc1�/�

mice

One hallmark presentation in NPC human disease is an enlargedliver and/or spleen [1]. To analyze to what extent the Npc1�/�

mouse model mimics human disease pathology, we measured thesize of the livers, spleens, and brains of 4-week-old mice (Table1). We found that relative liver size was significantly increased inNpc1�/� mice from both genetic backgrounds in comparison tomatched age controls (Table 1). Interestingly, C57BL/6J-Npc1�/�

mice presented a decrease in spleen size in comparison to wild typecontrols, which suggests dysfunction of this organ. One interestingand unexpected finding was that C57BL/6J-Npc1�/� mice had a rel-atively larger brain sizes in comparison to C57BL/6J-Npc1+/+ mice.

NPC disease is a lipidosis that affects multiple organs. The pri-mary lipid that accumulates is cholesterol; however, the build-upof other lipids, such as phospholipids, has been described [13,23].To corroborate these prior findings, we measured the lipid contentof the brain, liver, and spleen of 4-week-old mice. No change inbrain cholesterol content was observed at this age (Table 2). The li-ver and spleen cholesterol content was increased in Npc1�/� micefrom both genetic backgrounds in comparison to wild type, witha larger relative increase in C57BL6/J Npc1�/� mice (Table 2). Final-ly, we evaluated the brain, liver, and spleen phospholipid content.BALB/c-Npc1�/� and C57BL/6J-Npc1�/� mice showed no differencesin brain or liver phospholipid content, whereas a 5-fold difference

C57BL6/JNpc1 -/-

BALB/cNpc1 -/-

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100

20 40 60 80

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viva

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18 28 38 48 58 68 78 88

age (days)

wei

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C57BL6/JNpc1-/- BALB/cNpc1-/-

A

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Fig. 1. The survival and body weight of BALB/c- and C57BL/6J-Npc1�/� mice. (A)NPC mouse survival from the BALB/c and C57BL/6J genetic backgrounds. (B) Weightgain in NPC mice from the BALB/c and C57BL/6J genetic backgrounds. Five to sixanimals per group were analyzed.

Table 1Brain, liver and spleen relative weights of BALB/c and C57BL/6J wild-type and NPC1mutant mice.

Mice Body weight (g) Brain Liver

BALB/cWT 17.63 ± 2.82 2.14 ± 0.30 5.6 ± 0.5 0.71 ± 0.15NPC 13.32 ± 3.24a 2.50 ± 0.58 6.6 ± 0.4 0.75 ± 0.19

C57BL/6JWT 16.84 ± 1.68b 2.29 ± 0.19 5.6 ± 0.4 0.60 ± 0.25NPC 6.93 ± 2.08a,b,c 4.47 ± 0.92a,b,c 6.3 ± 0.7 0.31 ± 0.14a,b,c

Values are expressed as mean ± SD. Brain, liver and spleen weights are expressed asthe percentage of body weight.

a Significantly different from BALB/c wild-type mice.b Significantly different from BALB/c-NPC1 mutant mice.c Significantly different from C57BL/6J wild-type mice. WT, wild-type; NPC, NPC1

mutant.

402 J. Parra et al. / Biochemical and Biophysical Research Communications 413 (2011) 400–406

was observed in the spleens of C57BL/6J-Npc1�/� mice in compar-ison to wild type. This significant increase in lipid content in thespleens of C57BL/6J-Npc1�/� mice suggests that spleen damagedue to lipid build-up is an important cause of the phenotypic dif-ferences between the mouse strains.

3.3. The histopathology of BALB/c and C57BL/6J NPC1 mutants

Next, we analyzed the brain, liver and spleen histology of4-week-old Npc1�/� mice. Brains from both genetic backgrounds

showed the same features: enlarged and swollen neurons with lar-ger polar prolongations that appear similar to meganeurites. Nodifferences between the backgrounds were noted (Fig. 2A–D).

Liver samples from Npc1�/� mice from both genetic back-grounds showed a large number of single and clustered foamy cellsnear the sinusoids, periportal space, and the central vein. No differ-ence between the genetic backgrounds was observed (Fig. 2E–H).

Spleens from C57BL/6J-Npc1�/�mice showed severe histologicaland morphological abnormalities, including a large number of foa-my cells. The white pulp showed large clusters of foamy cells in thegerminal center and marginal area of the splenic nodule. The redpulp showed alterations in the organization and morphology ofthe splenic cords (Fig. 2I–P). In both pulps, but mainly in the redpulp of C57BL/6J-Npc1�/�, the magnitude of foamy cell infiltrationwas so great that disruption of the cellular architecture was fre-quently observed, whereas BALB/c-Npc1�/� mice showed a moreconserved splenic nodule and red pulp architecture.

One hallmark of the juvenile form of NPC disease that is mim-icked by the BALB/c-Npc1�/� mouse model is the progressive lossof cerebellar Purkinje cells [17,18]. We analyzed the presence ofcerebellar Purkinje cells in 4-week-old Npc1�/� mice from both ge-netic backgrounds by calbindin staining. We did not find Purkinjecell degeneration at this age in either of the genetic backgrounds(Fig. 3). In agreement with this result, we found a similar magni-tude of free cholesterol accumulation, predominantly in the Pur-kinje cells, by filipin staining analysis in both mutants (Fig. 3).

These results show that Npc1�/� mice are differentially affectedby splenic disease depending on their genetic background. Npc1�/�

mice from the the C57BL/6J genetic background showed strikingspleen damage at early ages, whereas Npc1�/� mice from theC57BL/6J genetic background showed only minor spleen alterations.

3.4. Blood cells analysis in BALB/c and C57BL/6J NPC1 mutants

Due to the severity of the spleen damage observed in the C57BL/6J-Npc1�/� mice, we decided to analyze the morphology and quan-tity of a blood cell series. Microscopic analysis of the blood cells re-vealed dramatic differences between Npc1�/� mice from the BALB/c and C57BL/6J genetic backgrounds. Red cells from C57BL/6J-Npc1�/� mice showed a dark center surrounded by a white ringand a dark outer layer. BALB/c-Npc1�/� red cells showed no mor-phological alterations in comparison to C57BL/6J- and BALB/c-Npc1+/+ red cells. In addition, the red series from C57BL/6J-Npc1�/

� mice also showed an increased number of abnormal circulatingerythrocytes. Unstained ghost cells (Fig. 4) and irregular erythro-cytes were detectable, both signs of degenerating cells. In agree-ment with this result, C57BL/6J-Npc1�/� mouse blood cellspresent with a lower mean corpuscular hemoglobin concentrationin comparison to the other three groups (Table 3).

The peripheral blood smears from C57BL/6J-Npc1�/� miceshowed significant atypias in the white series, including neutrophilhypersegmentation and cytoplasmic granulations (Fig. 4). Further-more, C57BL/6J-Npc1�/� mice showed smaller amounts of lympho-cytes than controls, revealing a relative and absolute lymphopenia(Table 3).

No differences in platelets were found between Npc1+/+ andNpc1�/� mice regardless of the genetic background (Table 3).

In conclusion, these results suggest an important effect of thegenetic background on the peripheral blood cells of Npc1�/� mice.

4. Discussion

This study strongly suggests that genetic background dramati-cally changes the progression of NPC disease in mice. While BALB/c-Npc1�/� mice are an excellent model for the study of juvenile

Table 2Cholesterol and phospholipid content of brain, liver and spleen of BALB/c and C57BL/6J wild-type and NPC1 mutant mice.

Cholesterol Phospholipid

Brain (mg/g of organ) Liver (mg/g of organ) Spleen (mg/g of organ) Brain (mg/g of organ) Liver (mg/g of organ) Spleen(mg/g of organ)

BALB/cWT 11.3 ± 1.2 2.3 ± 0.9 0.90 ± 0.08 41.1 ± 1.6 27.0 ± 7.0 18.5 ± 5.1NPC 11.4 ± 1.4 19.4 ± 6.3a 6.95 ± 1.75a 47.0 ± 5.3 47.7 ± 6.1a 20.5 ± 2.5NPC/WT 1.0 8.4 7.7 1.1 1.8 1.1

C57BL/6JWT 10.8 ± 1.0 1.1 ± 0.5 1.70 ± 0.10 46.5 ± 1.6 23.8 ± 3.6 14.1 ± 3.4NPC 13.5 ± 3.2 13.5 ± 0.9a,b 26.67 ± 6.77a,b,c 47.5 ± 7.1 47.5 ± 7.1a,b 72.1 ± 9.3a,b,c

NPC/WT 1.3 12.3 13.3 1.1 2.0 5.1

Values are expressed as mean ± SD. Measurements were performed in 6 mice in each experimental group.a Significantly different from BALB/c wild-type mice.b Significantly different from C57BL/6J wild-type mice.c Significantly different from BALB/c-NPC1 mutant mice. WT, wild-type; NPC, NPC1 mutant.

BRAIN

LIVER

SPLEEN

WH

ITE PU

LPR

ED PU

LP

BALB/c WT BALB/c NPC C57BL/6J WT C57BL/6J NPC

Fig. 2. Histological analysis of the brains, livers and spleens of BALB/c- and C57BL/6J-Npc1�/� mice. All of the analyses were performed in 28-day-old mice. Hematoxylin andeosin staining of control and NPC mice from the BALB/c and C57BL/6J genetic backgrounds is shown. Images were taken with a 40X objective. (A–D) WT and NPC brains fromthe BALB/c and C57BL/6J genetic backgrounds are. 40� magnification; single arrows indicate swollen neurons with polar elongations. (E–H) WT and NPC livers from BALB/cand C57BL/6J genetic backgrounds. Single arrows indicate terminal sinusoids in the central vein; double arrows indicate foam cells clusters; dashed circles indicate the lumenof the central vein. (I–L) WT and NPC white pulp spleens from the BALB/c and C57BL/6J genetic backgrounds. Single arrows indicate the central artery; double arrows indicatefoam cells clusters. (M–P) WT and NPC red pulp spleens from the BALB/c and C57BL/6J genetic backgrounds. 10�magnifications; double arrows indicate foam cells clusters.Scale bars are 20 lm in A–D; Scale bars are 50 lm in E–P.

J. Parra et al. / Biochemical and Biophysical Research Communications 413 (2011) 400–406 403

NPC disease, C57BL/6J-Npc1�/� mice model some features of infan-tile NPC disease.

Major differences between Npc1�/� in both backgrounds werefound in the spleen. Both mutants accumulated cholesterol in this

organ; however, C57BL/6J-Npc1�/� had a higher spleen cholesterolcontent than BALB/c-Npc1�/� mice at 4 weeks old. Althoughsplenomegaly is one of the hallmarks of NPC disease in mice, thespleen weight at 4 weeks old was reduced in affected C57BL/6J

CEREBELLUM

BALB/c WT BALB/c NPC C57BL/6J WT C57BL/6J NPC

CALBIN

DIN

FILIPIN

Fig. 3. Purkinje cell survival and cholesterol accumulation in BALB/c- and C57BL/6J-Npc1�/� mice. 28-day-old WT and NPC cerebella from BALB/c and C57BL/6J geneticbackground mice were analyzed for calbindin immunoreactivity by immunohistochemistry (20�magnifications for A–D) and unsterified cholesterol accumulation by filipinstaining (40� magnification for E–H). Scale bars are 50 lm.

BLOOD CELLS

WT NPC

BALB/cC

57BL/6J

C57BL/6J

CELLS AM

PLIFICA TIO

N

Fig. 4. Peripheral blood smear (20�). 28-day-old WT and NPC blood cells from the BALB/c and C57BL/6J genetic backgrounds are shown in A–D. Scale bars are 7 lm. Details ofthe blood abnormalities in C57BL/6J NPC mice are shown in E–H. E and F show white cell atypias (cytoplasmic granulations and neutrophil hypersegmentation, respectively).G and H show red cell abnormalities (dark centers and a dark outer layer cell and ghost erythrocytes, respectively). Single arrows indicate the target cell; double arrowsindicate ghost erythrocytes. Scale bars are 7 lm.

404 J. Parra et al. / Biochemical and Biophysical Research Communications 413 (2011) 400–406

mice and showed dramatic alterations in the red pulp architecture.These results suggest rapid spleen damage with probable severedysfunction of this organ.

In addition to spleen abnormalities, blood analysis revealed dif-ferences between both mice groups. C57BL/6J Npc1�/� mice

showed red cell abnormalities such as abundant ghost erythro-cytes, suggesting an inability of the spleen to remove them fromcirculation. Cells resembling reticulocytes were present in theblood of the C57BL/6J Npc1�/�, but not in the BALB/c Npc1�/� mice,indicating an accelerated eritropoyesis. In addition, we found a

Table 3Blood cell count of BALB/c and C57BL/6J wild-type and NPC1 mutant mice.

Red series White series Platelets(106/mm3)

RBC (106/mm3) Hem (gr/dL) MCHC (%) WBC LYM (103/mm3) NT

BALB/cWT 6.2 ± 0.36 31.8 ± 1.06 30.2 ± 1.70 3.3 ± 0.35 2.55 ± 0.64 0.5 ± 0.2 635.0 ± 125.9NPC 5.6 ± 2.46 28.9 ± 13.7 30.3 ± 1.48 2.5 ± 1.27 2.03 ± 1.36 0.4 ± 0.1 442.5 ± 217.8NPC/WT 0.9 0.9 1.0 0.8 0.8 0.8 0.7

C57BL/6JWT 6.2 ± 0.51 30.7 ± 2.88 34.5 ± 0.85 6.1 ± 2.11a,b,c 5.77 ± 2.14a,b,c 0.3 ± 0.2 709.5 ± 60.1NPC 6.5 ± 0.33 32.2 ± 1.33 17.8 ± 2.03a,b,c 2.4 ± 1.16 1.52 ± 0.22b 0.1 ± 0.1 690.5 ± 60.0NPC/WT 1.0 1.0 0.5 0.4 0.3 0.3 1.0

Values are expressed as mean ± SD. Measurements were performed in 5 mice in each experimental group.a Significantly different from BALB/c wild-type mice.b Significantly different from C57BL/6J wild-type mice.c Significantly different from BALB/c-NPC1 mutant mice. WT, wild-type; NPC, NPC1 mutant; RBC, red blood cells; Hem, hematocrit (packed cell volume); MCHC, mean

corpuscular hemoglobin concentration; WBC, white blood cells; LYM, lymphocytes; NT, neutrophils.

J. Parra et al. / Biochemical and Biophysical Research Communications 413 (2011) 400–406 405

decreased mean corpuscular hemoglobin concentration, indicatingthat the concentration of hemoglobin in a given volume of packedred blood cells is low, stemming from either a decrease in the abso-lute amount of circulating hemoglobin or an increase in red cellmass without a corresponding increase hemoglobin content. Thisobservation reinforces the previous idea of a failure in red bloodcell function, even with normal cell counts. This condition wouldhave a similar impact as iron deficiency anemia, which has beenpreviously reported in infantile NPC patients [24,25]. Indeed, thesehematological alterations suggest poor tissue perfusion. A decreasein effective oxygenation could promote anaerobic metabolism,generate oxidative stress products in the short term, and lead toa poor prognosis for the disease.

The white series from the C57BL/6J-Npc1�/� mice demonstrateda lower quantity of lymphocytes than Npc1+/+ and initiated a lym-phopenia. This result suggests that the Npc1 mutation impacts theimmune system of these mice. This deficiency in acquired immu-nity, in addition to structural damage of lymphoid organs (liverand spleen), could make these mice especially susceptible to infec-tion, as are NPC patients [27,28]. In addition, we found significantatypias in the peripheral blood smear, including hypersegmenta-tion and cytoplasmic granulations in neutrophils, which suggestshyperactivity of the granular cells and is consistent with a basalinflammatory state that can aggravate the deficit in red blood cellproduction [29].

These alterations in the C57BL/6J-Npc1�/� mouse peripheralblood smear and cell count is compatible with a splenectomized-like situation, with dysfunctional red blood cells in circulatingblood (ghost cells), a normal platelet count, and an immunocom-promised state that is consistent with an inability of the spleento clear dysfunctional cells from circulation and lymphoid organdamage.

To determine the relevance of the brain pathology of BALB/c-Npc1�/� mice, Loftus et al. created a transgenic mouse in whichthey restored NPC1 in the CNS [26]. These mice showed a normallifespan and lack of neurodegeneration, demonstrating thatBALB/c-Npc1�/� mice die from brain disease. In addition, Lopezet al. showed that rescuing NPC1 in neurons is sufficient to extendthe lifespan of FVB-Npc1�/� mice [30]. Our results show thatC57BL/6J-Npc1�/� present dramatic spleen pathology, which sug-gests that these mice do not die from brain disease. The underlyingmechanism and the genetic basis of this differential response andrelationship to lipid accumulation requires further study.

The relevance of NPC1-modifying genes has been described be-fore by Miyawaki et al. for the spm-npc1 mutation [12]. They dis-covered that when the spm mutation was transferred from theC57BL/6ksJ background to either the C57BL/6J or DBA/2J inbred

strain ground, the phenotype was modified, hepatosplenomegalywas decreased and lifespan was shortened [30]. Furthermore, amodifier of this Npc1 mutation was mapped to mouse chromosome19 by linkage analysis of the severity of the NPC phenotype in aBALB/c and DBA/2J F2 cross. Further studies are required to narrowthe rather broad region identified on mouse chromosome 19 and todetermine if the same modifier acts on the severity of the Npc1�/�

phenotype obtained in this study by transferring the Npc1-npcnih

mutation from the BALB/c background into the C57BL/6J inbredstrain.

NPC disease in humans is heterogeneous, and various pheno-types have been documented in infants to adults [1,9]. Althoughthe NPC1 genotype predicts the severity of the neurological courseto some degree, other features of the disease, such as perinatal liverinvolvement, seem to be independent of the NPC1 mutation [9]. Inthis context, studies in inbred mouse strains can help find modifi-ers of NPC disease progression.

In conclusion, our results show that genetic background has astrong influence on NPC disease expression from the Npc1-npcnih

mutation. Further studies are required to find modifier genes in-volved in these genetic background differences and their relevanceto human NPC disease.

Financial support

This work was supported by Fondo Nacional de Desarrollo Cientí-fico y Tecnológico (FONDECYT, Grants Nos. 1000567, 1030415 and1070622) and Dirección de Investigación, Facultad de Medicina,PUC (Programa Ayudante Alumno and Inmersión de Verano).

Acknowledgment

The authors thank the ARA Parseghian Research Foundation forsupporting research on Niemann–Pick type C disease.

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