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. Author manuscript; available in PMC: 2011 Jul 1.
Published in final edited form as: Biochim Biophys Acta. 2010 May 2;1802(7-8):601–608. doi: 10.1016/j.bbadis.2010.04.006

Factors that affect postnatal bone growth retardation in the twitcher murine model of Krabbe disease

Miguel Agustin Contreras 1, William Louis Ries 2, Srinivasan Shanmugarajan 3, Gonzalo Arboleda 4,1, Inderjit Singh 5, Avtar Kaur Singh 6,*
PMCID: PMC2950787  NIHMSID: NIHMS203236  PMID: 20441793

Abstract

Krabbe disease is an inherited lysosomal disorder in which galactosylsphingosine (psychosine) accumulates mainly in the central nervous system. To gain insight into the possible mechanism(s) that may be participating in the inhibition of the postnatal somatic growth described in the animal model of this disease (twitcher mouse, twi), we studied their femora. This study reports that twi femora are smaller than of those of wild type (wt), and present with abnormality of marrow cellularity, bone deposition (osteoblastic function), and osteoclastic activity. Furthermore, lipidomic analysis indicates altered sphingolipid homeostasis, but without significant changes in the levels of sphingolipid-derived intermediates of cell death (ceramide) or the levels of the osteoclast-osteoblast coupling factor (sphingosine-1-phosphate). However, there was significant accumulation of psychosine in the femora of adult twi animals as compared to wt, without induction of tumor necrosis factor-alpha or interleukin-6. Analysis of insulin-like growth factor-1 (IGF-1) plasma levels, a liver secreted hormone known to play a role in bone growth, indicated a drastic reduction in twi animals when compared to wt. To identify the cause of the decrease, we examined the IGF-1 mRNA expression and protein levels in the liver. The results indicated a significant reduction of IGF-1 mRNA as well as protein levels in the liver from twi as compared to wt littermates. Our data suggest that a combination of endogenous (psychosine) and endocrine (IGF-1) factors play a role in the inhibition of postnatal bone growth in twi mice; and further suggest that derangements of liver function may be contributing, at least in part, to this alteration.

Keywords: Galactosylsphingosine, Insulin-like growth factor-1, Krabbe disease, lysosomal disorders, osteopenia, psychosine, twitcher mice

1. Introduction

The infantile form of Krabbe disease (Globoid cell leukodystrophy) is the most common, a severe, rapidly progressing, neurodegenerative and invariably fatal, inherited metabolic disease produced by mutations on the β-galactosylcerebrosidase gene, which expresses a lysosomal enzyme that participates in the degradation of several glycosphingolipids [1, 2]. As a consequence, galactosylsphingosine (psychosine, also a substrate of the enzyme [3]) accumulates in the nervous system [3] and peripheral organs [4]. The availability of a mutant mouse (twitcher mouse; twi), that is a morphologically, genetically and enzymatically authentic murine model of the human disease [5], has been a valuable tool to advance our understanding of the development/progression of the disease in the central nervous system. In the nervous system, accumulation of psychosine exerts a cytotoxic effect especially on myelin-producing cells (oligodendrocytes) [6], with further induction of myelin degeneration, astrocytic gliosis, axonal loss and presence of multinucleate giant (globoid) cells [1]. Psychosine-induced local inflammatory response in the nervous system (induction of proinflammatory cytokines and intermediates)[7–10] plays a role in the early apoptotic loss of oligodendrocytes [11, 12] observed in Krabbe patients [13, 14] and twitcher mice [15] brains.

Twitcher mouse develops clinical signs by day 20th, characterized by stunted growth, twitching and hind leg weakness; and the disease progresses to a near-terminal stage by day 40–45 [1]. The twitching sign is associated with the loss of oligodendrocytes [16–18]; however, the cause of decreased somatic growth is unclear. Multiple etiologies could underlie this growth defect; but alteration of hypothalamic-hypophysial axis function may play a causative role. Growth hormone (GH), secreted by the anterior pituitary into the circulation, has long been recognized as one of the principal factors that control postnatal growth [19]. GH acts as a promoter of skeletal long bone growth and development as well as chondrocyte maturation [20, 21], by receptor mediated induction of the transcription of genes whose expressed proteins are important in mediating its diverse effects on body growth and metabolism [22]. GH also stimulates the expression of hepatic and extrahepatic insulin-like growth factor-1 (IGF-1), that plays an essential GH-mediated anabolic role on skeletal and non skeletal tissues [23–28].

We have previously reported that psychosine accumulation mediates the induction of cytokines in twi liver, thus compromising some of its functions [29]. In the present report, , we studied the presence of endogenous (psychosine; Sph-1P) and humoral (IGF-1) factors that may be responsible for the stunted growth of femora of twi mice, to help identify possible mechanisms that may be playing a role in the postnatal somatic growth retardation of twi mouse. Our data indicate that: i) psychosine accumulates in femora of twi mice; ii) osteoclast function of twi femora (bone resorption) is enhanced; iii) tartrate resistant acid phosphatase (TRAP) activity is increased in the growth plate; iv) twi mouse femora present alteration of sphingolipid homeostasis, without signs of change in Sph-1P or a significant decrease in sphingomyelin or an increase in ceramide pool; v) liver secreted IGF-1 level is drastically reduced in twi mice plasma, and vi) IGF-1 transcripts and protein levels are significantly down regulated in twi liver. This study reports for the first time evidence that psychosine, acting either endogenously in the bone (local accumulation) or in conjunction with a disrupted endocrinal signal (IGF-1), are potential factors that mediate the alteration in bone growth, and hence the somatic growth of twi mice.

2. Material and methods

2.1. Animal model

All animal work for this study was performed under a protocol approved by the Institutional Animal Care and Use Committee. Twitcher heterozygote breeding pairs (C57BL/6J twi+/−) were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and twi(−/−) (twi) and age-matched littermates wild type wt(+/+) (wt) were used for this study at postnatal day 33–34. Wt and twi animals were sacrificed with an overdose of anesthetic and tissues were removed, frozen in liquid nitrogen and stored at −70°C.

2.2. Femora analysis of psychosine and sphingolipid levels by HPLC-mass spectrometry

For lipidomic analysis, bones (left and right femur) were defrosted in ice-cold phosphate buffered saline and the muscle tissue removed manually. Bones were weighed, enveloped in aluminum foil, frozen in liquid nitrogen and crushed in a mortar. Quantitative high pressure liquid chromatography-tandem mass spectrometry analysis of psychosine and bioactive sphingolipids were performed on organics solvent extracts derived from wt and twi crushed bones, at the University Lipidomics Core facility [30].

2.3 Calcein bone labeling

To define abnormal bone growth in the twi mouse, a modified histomorphometric analysis was performed using double labeling with calcein as a marker [31]. Adult mice were given two intraperitoneal injections of calcein 5 days apart (postnatal days 26 and 31). Calcein was prepared in a 2% solution of sodium bicarbonate, and injected at a dose of 20 mg/kg of body weight. Animals were killed 2 days after the last injection (post natal day 33). Femora were isolated and calcein was assessed in undecalcified bones embedded in methylmethacrylate. Sections 4–6 µm thick (Leica RM 2155 rotary microtome; Leica Microsystems, Ontario, Canada) were examined under a fluorescence microscope (Olympus BX-60; excitation at 485 nm and emission at 510 nm), and the acquired images were rendered using Adobe Photoshop 7.0.

2.4. Histology and tartrate-resistant acid phosphatase (TRAP) staining analysis

Wild-type and twitcher mice bone specimens were fixed in 4% paraformaldehyde and dehydrated with serial changes in 70–100% ethanol. The specimens were embedded in methylmethacrylate and serial 4–6 µm sections were stained by Goldner’s trichrome method for light microscopy analysis [32] or stained for TRAP activity using a histochemical kit (Sigma-Aldrich) as described elsewhere [33].

2.5. Plasma and liver analysis of Insulin-like Growth Factor-1

Blood from wt and twi mice was collected in EDTA-K containing vaccutainer tubes (BD Biosciences, Franklin Lakes, NJ), and the plasma was separated by centrifugation at 900×g for 10min (Allegra X-15R centrifuge, Beckman-Coulter, Fullerton, CA) and stored at −70°C. Levels of IGF-1 were determined using an ELISA kit (Quantikine, mouse IGF-1 immunoassay according to manufacturer instructions (R&D Systems, Minneapolis, MN).

2.6. Bone analysis of cytokines (Tumor Necrosis Factor-alpha and Interleukin-6)

Expression of cytokines (TNF-α and IL-6) was detected by ELISA in the supernatant of lysis buffer bones’ extracts (RIPA buffer, Thermo Scientific Inc., Waltham, MA). Bones were individually processed as indicated for the mass spectrometric analysis and extracted with 120 uL of lysis buffer. ELISA analysis was performed according to manufactured instructions (TNF-α: eBioscience, San Diego, CA; IL-6: BD Biosciences, San Jose, CA) using 50 uL of bone’s extract. Plates’ readings were performed in a Spectra Max 190 ELISA reader (Molecular Devices, Sunnyvale, CA).

2.7. Quantitative polymerase chain reaction

Liver total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA) and the purified RNA used for the synthesis of cDNA. Single-strand cDNA was synthesized from 2µg of total RNA using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc, Hercules, CA) according to manufacturer’s instructions. qPCR was conducted using iCycler (iCycler iQ, Bio-Rad), specific primers for mouse IGF-1 (Cat. number PPM03387E; SABiosciences, Frederick, MD) and iQ SYBR Green Supermix (Bio-Rad). Thermal cycling conditions were as follows: activation of DNA polymerase at 95°C for 10 min, followed by 40 cycles of amplification at 95°C for 30s and 60°C for 30s. The expressions of target genes were normalized with respect to the house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All samples were run in duplicate. Statistical analysis was performed by student-t test (Graph Path InStat3, GraphPath Software Inc., San Diego, CA) and the difference at p <0.05 was considered statistically significant.

3. Results

3.1 Retarded growth of long bones (femora) of Twitcher mouse and Galactosyl-sphingosine (psychosine) accumulation

Postnatal growth curves show that twi mouse body weight is severely inhibited by day 20 (Fig. 1A), as reported by several laboratories [34–36]. Taking long bones (femora) as representatives of the animals’ growth, we analyzed their physical differences between wt and twi mice. The analysis of femora of wt and twi at post natal day 32–33 indicated that twi bones have smaller size and weighed 27% less than wt mice bones (Fig. 1B). Furthermore, determination of psychosine levels by mass spectrometry indicated that psychosine accumulates in twi bones of adults animals (33 days old), at levels 14 fold higher than the levels found in wt (Table 1).

Figure 1.

Figure 1

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Growth curves and differences in femora size and weight between wild type and twitcher mice. Significant differences in body weight (A), and length and weight (B) between femora from wild type and litters mate twitcher mice is evident in adult animals. Graph represents the average ± SD for n = 6–7 animals per group. ***: Asterisks denote significant statistical differences from wild type littermates (p < 0.0001). WT: wild type mice; TW: Twitcher mice.

Table 1.

Level of galactosylsphingosine (psychosine) in wild type and twitcher mice femora

Femoraa
(pmoles/g tissue)
Wild type 13 ± 1
Twitcher 187 ± 57**
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(a)

Samples represent Mean ± SD of psychosine concentrations found in femora from wild type (n = 3 sets) and twitcher (n = 4 sets) mice, determined at postnatal day 32–33. (Set includes the left and the right femur from each animal).

**

p < 0.004

3.2 Histomorphologic analysis of Twitcher femora

Examination of stained wt and twi femora under light microscopy revealed differences in cortical bone thickness, cellular organization (isogenous groups) and width of the growth plate, between wt and twi animals (Fig. 2A, B). The metaphyseal region of twi bones also showed marked structural differences, with reduction of trabecular bone (stained in green) mainly in the region of the secondary spongiosa (Fig. 2C, D). Overall, there was a marked decrease in cortical bone thickness and the number of trabeculae, indicating the twi mouse shows an osteopenic phenotype.

Figure 2.

Figure 2

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Morphologic differences between the femur from wild type and twitcher mice. Light microscopic study of Goldner’s trichrome stained sections derived from wild type (A, C) and twitcher (B, D) mouse bone identified differences in thickness of green-stained cortical bone (CB), organization and morphology of the chondrocytes and width of the growth plate (GP) and reduction of green-stained trabecular bone (TB) (A, B: 40× magnification; line represents 2 mm; C, D: 60× magnification, line represents 200 µm).

3.3. Bone growth/remodeling functions (osteoblastic deposition and osteoclastic resorption) in femora of wild type and twitcher mice

To gain insight into the possible cause of reduced bone in twi femora, the bone-forming capacity of osteoblasts was examined by the deposition of calcein. The calcein labeling along the cortical bone (seen as two layers of florescent green lines under the fluorescence microscopic analysis) showed no difference between wt and twi (Fig. 3A, B), indicating no differences in the mineral apposition rate. However, there was a substantial reduction of bone deposition (green fluorescence) in the metaphyseal and epiphyseal region of twi femora (Fig. 3A, B). To evaluate whether enhanced osteoclastic activity is the cause of the observed reduced calcein deposition in both regions, we performed histochemical analysis for tartrate-resistant acid phosphatase (TRAP) activity. TRAP-positive osteoclasts were identified along the base of the growth plate and most of the trabeculae and on the endosteal surface of the diaphysis in both wt and twi mice bone showed activity (Fig. 3C, D). However, TRAP staining was stronger in twi bone, and included the narrow growth plate of the twi femur. These data indicate that psychosine-induced alterations in twi bone preferentially affects the function of osteoclasts and suggests that enhanced osteoclastic resorption may be playing a role in the bone growth retardation observed in twi animals.

Figure 3.

Figure 3

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Bone deposition (osteoblasts activity) and resorption (osteoclasts activity) functions in the femora of twitcher mice. Osteoblasts function in wild type (A) and twitcher (B) was determined by double calcein labels (green fluorescence) during new bone formation/remodeling. Osteoclasts function, as TRAP-positive osteoclasts (red) can be seen along the base of the growth plate as well as on most of the trabeculae (TB) and on the endosteal surface of the cortex in both wild type (C) and twitcher (D) mice. Note the drastic reduction of labeling in twitcher mouse trabeculae (trabecular bone, TB) (40× magnification; line represents 2 mm)(B) and the intense TRAP activity of the growth plate in twitcher mice (D). TRAP assay was done as indicated in Material and methods (40× magnification).

3.4. Sphingolipid homeostasis and cytokine markers in femora of twitcher mouse

To evaluate possible changes in the osteoclast-osteoblast coupling factor sphingosine-1 phosphate (Sph-1P)[37] and to better understand the cause of reduced cellularity reported in twi bone marrow [36], we studied the levels of sphingolipid in femora. The analysis of the wt and twi femora by mass spectrometry indicated a significant increase (1.3 fold) in the total sphingomyelin (SM) pool found in the twi (341 ± 10 nmoles/g) when compared to wt bones (263 ± 11 nmoles/g; p < 0.0001)(Fig. 4A). The observed increase is mainly a contribution of the saturated species C16:0-SM (1.3 fold), C18:0-SM (1.2 fold), C20:0-SM (1.4 fold), C22:0-SM (1.4 fold), and C24:0-SM (1.2 fold); and of the unsaturated species C22:1-SM (1.4 fold), C24:1-SM (1.4 fold) and C26:1-SM (1.1 fold) (Table 2). On the other hand, the analysis of the ceramide (CM) pool did not indicate a significant difference between wt and twi bones (Fig. 4A, B). However, significant changes were observed in the individual saturated species C20:0-CM (0.7 fold) and C22:0-CM (1.4 fold), DHC16:0 (0.8 fold), Sph (1.4 fold), and the unsaturated species C24:1-CM (1.3 fold) (Table 3; Fig. 4B). Interestingly, the most abundant species (C24:1) in SM as well as in CM was significantly elevated in both pools; and the dihydro-C16:0-CM (DHC16:0; a de novo CM synthesis indicator [38]) was significantly decreased. Taking into account that the changes observed in the total SM pool and SM species represent an increase in concentrations in twi as compared with wt, and minor variations in some CM saturated and unsaturated species, these data suggest that psychosine accumulation in twi bones is interfering with SM and CM turnover.

Figure 4.

Figure 4

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Sphingolipid concentration in wild type and twitcher femora. The levels of sphingomyelin (SM) and ceramide (CM) (A); sphingosine (Sph) and sphingosine-1-phosphate (Sph-1P) (B) fractions were determined by HPLC-mass spectrometry in organic extracts from pairs of femora of wild type (white bars) and twitcher mice (black bars), as indicated in Materials and methods. Values in (A) were obtained from the summary of the individual SM and CM fractions (including saturated and unsaturated fatty acids fractions). Graph represent the average ± SD for n = 4 samples per group. Asterisks denote significant statistical differences from wild type littermates (*: p < 0.01; **: p < 0.0001).

Table 2.

Determination of the sphingomyelin fractions composition in wild type and twitcher femora

Sphingomyelin fractionsa
(nmoles/g tissue)
C14:0 C16:0 C18:0 C20:0 C22:0 C24:0 C26:0 C18:1 C20:1 C22:1 C24:1 C26:1
Wild type 1.07 ± 0.05 110 ± 4 39 ± 3 4.7 ± 0.5 14 ± 2 36 ± 2 0.35 ± 0.05 2.9 ± 0.3 0.36 ± 0.07 2.6 ± 0.4 52 ± 5 0.75 ± 0.04
Twitcher 1.23 ± 0.17 143 ± 6 48 ± 2 6.5 ± 0.6 19 ± 2 42 ± 3 0.33 ± 0.05 3.2 ± 0.3 0.44 ± 0.06 3.6 ± 0.4 73 ± 4 0.85 ± 0.05
p < ns 0.0001 0.001 0.001 0.002 0.005 ns ns ns 0.005 0.0002 0.01
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(a)

Individual sphyngomyelin fractions were determined by mass spectrometry in defleshed bone (wet weight) organic extracts. Values represent the average ± SD for n = 5 sets per group. p <: Statistical significance as indicated, versus wild type; ns: not significant.

Table 3.

Determination of the ceramide fractions composition in wild type and twitcher femora

Ceramide fractionsa
(nmoles/g tissue)
C14:0 C16:0 C18:0 C20:0 C22:0 C24:0 C26:0 C18:1 C20:1 C20:4 C22:1 C24:1 C26:1 DHC16:0
Wild type 6.2 ± 3.4 32 ± 8 10 ± 2 10.4 ± 2.1 7.5 ± 0.8 33 ± 4 0.13 ± 0.04 4.2 ± 0.3 2.8 ± 0.4 0.07 ± 0.02 3.7 ± 0.5 38 ± 5 0.36 ± 0.06 22 ± 3
Twitcher 5.0 ± 2.2 34 ± 4 10 ± 1 7.5 ± 1.1 10.3 ± 0.5 35 ± 5 0.11 ± 0.06 3.7 ± 0.6 2.3 ± 0.4 0.04 ± 0.01 4.0 ± 0.4 51 ± 4 0.32 ± 0.07 18 ± 1
p < ns ns ns 0.03 0.0002 ns ns ns ns ns ns 0.001 ns 0.03
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(a)

Individual ceramide fractions were determined by mass spectrometry in defleshed bone (wet weight) organic extracts. Values represent the average ± SD for n = 5 sets per group. p <: Statistical significance as indicated, versus wild type; ns: not significant.

To rule out a possible psychosine-induced inflammatory process as demonstrated in the brains and livers of twitcher mice [8, 29], we determined the bone’s levels of the cytokines TNF-α and IL-6 (Table 4). Because the cytokine analysis did not indicate a significant difference between wild type and twitcher mice (Table 4), the results suggest that psychosine is not exerting its effect by inducing the expression of mediators of inflammation.

Table 4.

Level of cytokines (TNF-α and IL-6) in wild type and twitcher mice femora

TNF-α IL-6
(pmoles / g tissue)a
Wild type 86 ± 56 152 ± 79
Twitcher 112 ± 76 149 ± 63
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(a)

Samples represent Mean ± SD of cytokines concentrations (tumor necrosis factor-alpha, TNF-α; Interleukin-6, IL-6) found in femora from wild type (n = 4 sets) and twitcher (n = 5 sets) mice, determined at postnatal day 33–35. (Set includes the left and the right femur from each animal).

3.5 Insulin-like growth factor-1 (IGF-1) levels in plasma and mRNA expression and protein levels in liver

IGF-1 is a GH-dependent growth factor produced mainly by the liver, and it is known to regulate growth and bone remodeling [39]. ELISA analysis of plasma levels of IGF-1 in wt and twi animals indicated that IGF-1 levels were significantly reduced by 37.6% in twi animals as compared to wt (234.4 ± 26.4 vs 148.7 ± 20.5 ng/ml in wt and twi respectively; p < 0.002)(Fig. 5), and decreases as the disease progresses (Fig. S1A, B). Because plasma circulating IGF-1 is of hepatic origin and liver is also affected by psychosine induced-disease in twi mice [29], we investigated the mRNA expression and protein levels in liver of wt and twi mice. The hepatic expression of IGF-1 mRNA was down regulated by 59.3% in adult animals (1.9 ± 0.4 in wt vs. 0.8 ± 0.3 folds in twi; p < 0.005)(Fig. 6A), whereas IGF-1 protein levels were decreased by 60.4% (1.7 ± 0.2 in wt vs. 0.7 ± 0.3 ng/mg of protein in twi; p < 0.0001)(Fig. 6B) in twi liver as compared with wt. This result indicated a strong alteration of the liver functions in twi animals.

Figure 5.

Figure 5

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Insulin-like growth factor-1 levels in the plasma of twitcher mice. Plasma levels of IGF-1 in wild type and twitcher mice were determined by ELISA assay at postnatal day 33. Graph represents the average ± SD for n = 4 animals per group. Asterisks denote significant statistical differences from wild type littermates (**: p < 0.002).

Figure 6.

Figure 6

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Expression and protein level of insulin-like growth factor-1 in twitcher mice liver. Wild type and twitcher mouse IGF-1 mRNA expression were determined by quantitative PCR (A), and IGF-1 protein levels in liver homogenate soluble fractions were determined by ELISA assay (B), as described in Materials and Methods. Graph represents the average ± SD for the number of animals indicated per group. Analysis was performed in mice 33–34 days old. **: p < 0.005 (n = 3); ***: p < 0.0001 (n = 6). Asterisks denote significant statistical differences from wild type littermates. WT: wild type mice; TW: Twitcher mice.

4. Discussion

Twitcher mouse is an animal model of the genetic disorder Globoid cell leukodystrophy (Krabbe disease) [5, 40, 41]. These animals develop clinical signs by day 20 (stunted growth, twitching, hind leg weakness), and they reach a near-terminal stage by day 40–45 [1]. While the twitching sign is associated with oligodendrocytes (myelin-forming cell) loss [16–18], the cause of twi mouse decreased growth is unclear. It is well established that psychosine accumulates in nervous tissue [3], and exerts an inflammatory response characterized by induction of proinflammatory cytokines, inducible nitric oxide synthase, oxidative imbalance and early oligodendrocyte cell death [8, 11–15, 42]. As the disease pathology progresses, the inflammatory process compromises further brain homeostasis, altering subcellular functions of peroxisomes [42, 43] and possibly the mitochondria [44, 45]. In parallel with the central nervous system events, psychosine also accumulates in non-nervous tissues/organs, affecting their homeostasis [29, 36]. Our data demonstrated that psychosine also accumulates in long bones (femora) of twitcher mice, where it may exert cytotoxic effects, thus supporting the notion that globoid cell leukodystrophy (Krabbe disease) is a generalized storage disease [4], and adding another tissue to the list of those that accumulate psychosine [29, 36, 46–48]. Such accumulation, however, does not compare to the levels found in twi liver or brain (3 and 27 folds higher than in bone, respectively) [29].

Many local [37, 49–51], humoral/endocrine [20, 21, 26] and central and peripheral nervous system-derived factors [39, 52] regulate the functions of the specialized cells such as osteoblasts (bone deposition) and osteoclasts (bone resorption) responsible for bone growth/remodeling [50]. Morphologic and functional analysis of wt and twi femora demonstrated reduced content of cortical and trabecular bone indicating that the twi mouse shows an osteopenic phenotype, possibly a consequence of enhanced osteoclastic bone resorption (TRAP activity) without much alteration of osteoblastic function (calcein deposition). These data indicate that psychosine-induced alterations in twi bone may uncouple bone deposition from bone resorption. It affects preferentially osteoclastic function and suggests that over time an enhanced osteoclastic resorption may be playing a role in the premature closure of the growth plate; and hence is a contributing factor to the reduction in bone growth in twi animals. Interestingly, in another animal model of lysosomal disease, mucopolyssacharidosis type VII (deficiency of the enzyme β-glucuronidase), there is metabolite accumulation mediated bone alteration by impairment of both osteoclastic and osteoblastic functions [53].

In most tissues, SM degradation (hence increase in CM levels) is associated with activation of stress response/cell death [54], and Sph-1P increase is associated with antiapoptotic/cell survival signal [55, 56]. In addition, in bone Sph-1P is a factor that regulates the osteoclast-osteoblast coupling during bone growth/remodeling [37, 51]. In general, our sphingolipid analysis resembles the changes reported for twi liver [29]; with insignificant changes in known apoptotic (C16:0-CM) or antiapoptotic (Sph-1P) sphingolipid fractions [38, 57]. However, an increase was found in total SM fraction, and in most of the SM and CM containing saturated-unsaturated fatty acids, including the most abundant species (C24:1) in SM and in CM. On the other hand, the dehydro-C16:0 (DHC16:0; an indicator of de novo CM synthesis [38]) was significantly decreased and Sph accumulated in twi mice. These data suggest that psychosine accumulation may interfere with SM degradation and CM synthesis in twi bones. Furthermore, the lipidomic analysis showing no significant changes on Sph-1P between wt and twi mice bone rules out that this sphingolipid may be playing a role in the osteoclast-osteoblast uncoupling [37]. These results suggest that the levels of psychosine may not reach a threshold that is enough to trigger stress response/cell death in twi bones. Consistent with these results, analysis of the inflammation marker TNF-α and IL-6 were found not to be altered, which strongly suggests that the psychosine-induced pathology in twitcher bones is not dependent or potentiated by the induction of mediators of inflammation, as demonstrated in the brain and liver of those animals [8, 29] or studies on cell culture [13]. On the other hand, psychosine accumulation may be interfering with cell signaling at the level of the receptor sites (lipids rafts), as suggested recently [58]. Future studies should be aimed to identify the cellular distribution of the accumulated psychosine in long bones.

Liver is the main organ that contributes to the blood circulating IGF-1 pool [27, 28, 59]; with IGF-1 playing an important role in bone physiology [25, 60, 61]. Taking into consideration that liver functions are compromised by psychosine-induced inflammation in twi animals [29], it was not surprising that mRNA expression and protein levels of IGF-1 were found to be down regulated in the liver of twi mice, supporting the drastic decrease in IGF-1 plasma levels observed in those mice. These results indicate an additional psychosine-mediated metabolic dysfunction in twi liver (synthesis of IGF-1), and suggest that reduction in IGF-1 plasma levels may be an additional factor contributing to the observed impaired balance (uncoupling) between osteoclast-osteoblast function, and hence to the bone growth stunting in twi mice. Studies performed in double gene disrupted mice (liver IGF-1-deficient and acid labile subunit (ALS) knockout) support our results [62]; dramatic decrease in IGF-1 levels in both animals is consistent with decreased bone growth. Indeed, the fewer trabeculae in the metaphysis is a sign of osteopenia; and the smaller growth plate in both cases is consistent with the narrowing of the growth plate observed with aging, which is associated with decrease in circulating and local production of IGF-1 [63–65]. Furthermore, additional factors such as the endogenous presence of cytokines (as seen in brain and liver)[8, 29] or alterations in the hypothalamic-pituitary axis in the nervous system [66], may also play a role in the derangements of bone growth. Recent studies have reported drastic changes in twi mice bone marrow functions [36].

Our data and that of others demonstrate the complexity ofthe development and progression of the psychosine-induced pathology in the nervous system [11–13, 36, 43] and in peripheral organs [29, 36], and highlight the importance of peripheral organs for the overall organism homeostasis [29]. Based on these observations, we hypothesize that progression of inflammation in the twi central nervous system could affect hormone secretion which may play a role in the homeostasis of peripheral organs, such as in bone growth regulation [67, 68].

In summary, we have identified two potential factors that may be affecting bone growth in twi mice. We report that psychosine accumulates in bones, where either maybe by itself as an endogenous factor or in conjunction with a decrease of plasma IGF-1 levels, preferentially impaired osteoclastic function, and hence stunted bone growth. Therefore, recent therapeutic treatments [69, 70] might consider the supplementation/induction of endocrinous/paracrine factors (i.e., GH-IGF-1). Combination therapy may prove to be of benefit for the central nervous system [71–73] and peripheral organs, and ultimately for the amelioration of the development and progression of the disease pathology in twi mice and Krabbe patients.

Supplementary Material

01

Figure S1. Postnatal differences in femora sizes and protein level of insulin-like growth factor-1 in plasma of wild type and twitcher mice. Postnatal femora from 10 to 45 days of age are shown in (A). White asterisks indicate differences in bones’ length between wild type and twitcher animals. Wild type (white bars) and twitcher mouse (black bars) IGF-1 protein levels in plasma were determined by ELISA assay at the age period indicated (B). Graph represents the average ± SD for the number of animals indicated per group. *: p < 0.02; ***: p < 0.0001 indicate significant statistical differences from wild type littermates. The number of animals in each age period were: postnatal days 20–25 (n = 6 WT, 6TW); 26–30 (n = 4WT, 7TW); 31–35 (n = 4WT, 6TW) and 36–40 (n = 7WT, 11TW). WT: wild type mice; TW: Twitcher mice.

Acknowledgements

The authors would like to thank Ms. Joyce Bryan, Dr. Narender Nath and the members of the Lipidomics Core Facility of the Medical University of South Carolina for the excellent technical assistance. This work was supported by Grants from the Extramural Research Facilities Program of the National Center for Research Resources (C06 RR018823 and C06 RR015455) and from the National Institutes of Health (NS-22576, NS-34741, NS-37766, NS-40810 and AG-25307).

Abbreviations

CM

ceramide

GH

growth hormone

IGF-1

insulin-like growth factor-1

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

qPCR

quantitative polymerase chain reaction

TRAP

tartrate resistant acid phosphatase

SM

sphingomyelin

Sph

sphingosine

Sph-1P

sphingosine-1-phosphate

Twi

twitcher

Wt

wild type

Footnotes

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Contributor Information

Miguel Agustin Contreras, Department of Pediatrics, Darby Children’s Research Institute, Medical University of South Carolina.

William Louis Ries, Department of Pediatrics, Darby Children’s Research Institute, Medical University of South Carolina.

Srinivasan Shanmugarajan, Department of Pediatrics, Darby Children’s Research Institute, Medical University of South Carolina.

Gonzalo Arboleda, Department of Pediatrics, Darby Children’s Research Institute, Medical University of South Carolina.

Inderjit Singh, Department of Pediatrics, Darby Children’s Research Institute, Medical University of South Carolina.

Avtar Kaur Singh, Department of Pathology and Laboratory Medicine, Ralph H. Johnson Veteran’s Administration Medical Center, Charleston, SC.

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Associated Data

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Supplementary Materials

01

Figure S1. Postnatal differences in femora sizes and protein level of insulin-like growth factor-1 in plasma of wild type and twitcher mice. Postnatal femora from 10 to 45 days of age are shown in (A). White asterisks indicate differences in bones’ length between wild type and twitcher animals. Wild type (white bars) and twitcher mouse (black bars) IGF-1 protein levels in plasma were determined by ELISA assay at the age period indicated (B). Graph represents the average ± SD for the number of animals indicated per group. *: p < 0.02; ***: p < 0.0001 indicate significant statistical differences from wild type littermates. The number of animals in each age period were: postnatal days 20–25 (n = 6 WT, 6TW); 26–30 (n = 4WT, 7TW); 31–35 (n = 4WT, 6TW) and 36–40 (n = 7WT, 11TW). WT: wild type mice; TW: Twitcher mice.

NIHMS203236-supplement-01.tif (9MB, tif)

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