Human Molecular Genetics, 2001, Vol. 10, No. 23 2709-2715
© 2001 Oxford University Press
Dramatic phenotypic improvement during pregnancy in a genetic leukodystrophy: estrogen appears to be a critical factor
1Neuroscience Center, 2Departments of Neurology and Psychiatry, 3Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599-7250, USA and 4INSERM U 189, Lyon-Sud School of Medicine and Fondation Gillet-Mérieux, Lyon-Sud Hospital, 69921 Oullins Cedex, France
Received August 1, 2001; Revised and Accepted September 9, 2001.
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ABSTRACT |
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Globoid cell leukodystrophy is one of the classical genetic leukodystrophies in humans. The typical infantile disease in man (Krabbe disease) is caused by deficiency of lysosomal galactosylceramidase. We recently generated a new mouse model of a late-onset, chronic form of the disease by inactivating saposin A, the essential activator of galactosylceramidase. The phenotypic features of saposin A–/– mice are qualitatively identical but milder than those of twitcher mice, which is caused by genetic galactosylceramidase deficiency. During intercrossing of saposin A–/– mice, we observed that affected females that are continually pregnant showed greatly improved neurological symptoms compared to affected females that do not experience pregnancy, or affected males. The pathological hallmark of globoid cell leukodystrophy, demyelination with infiltration of globoid cells, largely disappeared. The immune-related gene expression (MCP-1, TNF-
) was significantly down-regulated in the brain of pregnant saposin A–/– mice. In addition, we found intense expression of the estrogen receptors (ER and ERß) on the globoid cells, activated astrocytes and microglia in the demyelinating area of saposin A–/– mice. When saposin A–/– mice were subcutaneously implanted with time-release 17ß-estradiol (E2) pellets from 30 to 90 days, the pathology was vastly improved. These findings suggest that a higher level of estrogen during pregnancy is one of the important factors in the protective effect of pregnancy. While we should be cautious in extrapolating these observations in the mouse to human disease, the phenomenon is spectacularly dramatic and estrogen administration might be worth a consideration as a supplementary treatment for some chronic genetic leukodystrophies. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Globoid cell leukodystrophy (GLD) is one of the classical hereditary leukodystrophies (1). Genetic galactosylceramidase (GALC) (EC 3.2.1.46) deficiency was the only known cause of all GLD cases in man (Krabbe disease) and in several other mammalian species (1). Among them, the twitcher mouse has been widely used as a useful animal model (2). However, we have recently generated a new mouse model of late-onset, chronic GLD by introducing a mutation (C106F) in the saposin A domain of the sphingolipid activator protein gene (prosaposin) (3). The clinical, biochemical and pathological features are qualitatively identical with but milder than those seen in the twitcher mouse indicating that saposin A is an indispensable in vivo activator of GALC. Here we report that pregnancy dramatically alleviates the clinical and pathological phenotype of saposin A–/– mice and that estrogen administration can substantially duplicate the protective effects of pregnancy. The findings provide the first evidence that estrogen supplementation might be effective for chronic forms of genetic leukodystrophies.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Both saposin A–/– males and females are fertile, and females are capable of raising three litters of pups during the course of the disease. For this series of experiments, we used only offspring of saposin A–/– mating. All pups were genotyped soon after birth and then again randomly spot-checked when they were killed for tissue sampling. This design excluded any possible influence during pregnancy by normal saposin A carried by fetuses, since all fetuses were saposin A–/–. The mice described here were products of F2 generation, which still had a heterogeneous genetic background of 129 SV and C57BL6/J; subsequent observations with mice after backcrossing to C57BL6/J up to six times indicated that the genetic background is irrelevant to the observed phenomena.
Clinical phenotype
Affected females that were continually pregnant lived much longer with greatly improved neurological symptoms compared to affected females that did not experience pregnancy, or affected males (Fig. 1A). Saposin A–/– mice, which experienced pregnancy three times without interruption, appeared nearly normal even at 136 days, when male and virgin female saposin A–/– littermates showed severe gait disturbance and weight loss (Fig. 1B). Neurologically, saposin A–/– mice, which experienced pregnancy, retained their motor function much better than their littermates, although their walking pattern was flat-footed with a wider base and shorter strides than wild-type mice (Fig. 1C).
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Neuropathology
We compared the neuropathology of pregnant saposin A–/– mice with male and virgin female saposin A–/– littermates in 10 litters (the age range: 63–136 days). All pregnant saposin A–/– mice had much milder pathology. Most dramatic improvement was found in those mice that had experienced pregnancy three times without interruption. In saposin A–/– mice, peripheral nerves are abnormally firm and thick as typically seen in GLD (4). However, in pregnant saposin A–/– mice, nerves were supple and thin similar to those of wild-type mice (Fig. 2A) and the myelin sheaths were well preserved even at the terminal stage (Fig. 2B). In contrast to the extensive demyelination with numerous infiltrating periodic acid Schiff (PAS) stain-positive macrophages (‘globoid cells’) in male and virgin female saposin A–/– mice, pregnant females showed much less infiltration of PAS-positive macrophages in the central nervous system (CNS) (Fig. 2B).
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Brain psychosine
Psychosine (galactosylsphingosine) is highly cytotoxic and is one of the substrates of GALC. Experimental evidence has been accumulating in support of a hypothesis that psychosine accumulation may be primarily responsible for disappearance of the myelinating cells in GLD (5,6). Psychosine accumulates 10–20-fold in human patients with Krabbe disease as well as in GLD that occurs in other mammalian species (1,7–10). Reflecting the milder phenotype, the brain psychosine level in saposin A–/– mice is twice normal throughout the disease (3). Despite the dramatic clinical and pathological improvements, the brain psychosine levels in pregnant saposin A–/– mice showed no difference from those of affected littermates [wild-type, 32 ± 1 (n = 4); affected male, 62 ± 4 (n = 9); affected female without pregnancy, 61 ± 2 (n = 12); affected female with pregnancy, 60 ± 2 (n = 11); the age range: 81–150 days, expressed in pmol psychosine/mg protein, mean ± SD]. Thus, the mechanism with which pregnancy alleviates the phenotype is not through reduction in psychosine.
Cytokine/receptor expression
The abnormal metabolism of galactosylceramide and the accumulation of cytotoxic compound psychosine in the myelin-forming cells may be the two primary pathogenetic mechanisms in GLD. However, we earlier observed enhanced expression of a variety of cytokines in the brain of twitcher mice, suggesting the role of secondary inflammatory mechanisms in the pathogenesis of GLD (11). Therefore, we examined expression of IL-10, monocyte chemoattractant protein-1 (MCP-1), TNF- and TNF- receptor type 2 (TNFR2) in the brain of saposin A–/– mice by RT–PCR. These cytokines were generally up-regulated in the brain of saposin A–/– mice, whereas expression of MCP-1 and TNF- of the pregnant saposin A–/– mice was significantly lower than those of male saposin A–/– mice (P < 0.01, P < 0.05, respectively) (Fig. 3). TNFR2 expression was also higher in saposin A–/– mice, and male mice exhibited a significantly higher level relative to female mice both in saposin A–/– mice and wild-type mice (P < 0.05).
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Expression of estrogen receptors (ER
and ERß)To be a target for sex steroid hormones, cells must express the cognate receptor. We examined estrogen receptor (ER) expression in the CNS of saposin A–/– mice. We found intense nuclear and cytoplasmic expression of both ER
and ERß on the cells that showed morphological characteristics of macrophages, activated astrocytes or microglia (Fig. 4). Although ER is classically localized to nuclei, several studies also demonstrated extra-nuclear ER immunoreactivity (12–14).
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17ß-Estradiol (E2) implantation experiment
The high level of estrogen during pregnancy is one of the obvious candidates for the factors responsible for the protective effect of pregnancy. We subcutaneously implanted 3 mm pellets containing 15 mg 17ß-estradiol (E2) in a cholesterol matrix (Innovative Research of America, Sarasota, FL) to 30-day-old virgin female (n = 4) and male (n = 4) saposin A–/– mice. The pellets maintain the high pregnancy levels (5000–10 000 pg/ml) of estrogen over a period of 60 days (15–17). Control saposin A–/– mice (two females and two males) were sham-operated but received no estrogen pellet. For pathological evaluation, mice were killed after the 60 day estrogen treatment at around 90 days of age. In the natural course of the disease, saposin A–/– mice at 30 days are indistinguishable from wild-type mice and the GLD pathology is rarely detected and, at 90 days, the pathology is fully developed. Whereas clinical improvements were not as obvious as in natural pregnancy, pathologically, at
90 days, three out of four E2-treated female saposin A–/– mice, and two out of four E2-treated male saposin A–/– mice showed much better preservation of myelin and much less infiltration of the PAS-positive macrophages similar to those in continually pregnant saposin A–/– mice (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The experimental observations described above provide the first and strong evidence that pregnancy dramatically improves clinical and pathological phenotype of a late-onset chronic form of GLD and that estrogen supplementation substantially duplicates the protective effect of pregnancy, particularly on neuropathology. In late-onset human Krabbe disease, a few anecdotal cases have been mentioned that might be relevant to this study (18). Two patients are said to have improved with prednisone treatment. One patient worsened with steroids but improved upon administration of ACTH. One patient personally known to one of us developed her disease 3 months postpartum at 25 years (19). Since late-onset Krabbe disease is rare in humans, these cases must inevitably remain anecdotal. The saposin A–/– mouse provides an ideal experimental model to study this phenomenon in a well controlled manner, particularly because females can be pregnant with all fetuses in saposin A–/– genotype, thus excluding the possibility of beneficial effect of normal saposin A from fetuses.
In our model system, pregnancy did not affect the level of psychosine in the CNS. Clearly, pregnancy is not exerting its protective effect through reduction in psychosine. Psychosine is considered responsible for early disappearance of oligodendrocytes in the early onset form of GLD (5,6). Contrary to the twitcher mice, brain psychosine in untreated saposin A–/– mice is only twice normal. Our interpretation is that there are two distinct pathogenetic processes in GLD, infiltration of macrophages (globoid cells) due to undigestible galactosylceramide (20,21) and death of oligodendrocytes due to accumulation of toxic psychosine. Despite the recent report of the capacity of both galactosylsphingosine (psychosine) and glucosylsphingosine to generate multinucleated cells in a culture system (22) and psychosine receptor (23), only galactosylceramide, but not psychosine, has been shown to cause GLD-like macrophage infiltration when injected in the brain. In the infantile form of Krabbe disease and twitcher mice, both mechanisms are in operation, whereas in saposin A–/– mice, psychosine accumulation may not be high enough to cause detrimental effects on the oligodendrocytes and the macrophage infiltration may be primarily responsible for its pathogenesis. No information is available on brain psychosine levels in the adult form of human Krabbe disease.
One of the obvious candidates for active factor(s) in the mechanism of the protective effect of pregnancy is estrogen. Our results indicate that estrogen can mimic the pregnancy effect at least partially. The pathological improvements during pregnancy could be duplicated nearly completely by subcutaneous implantation of the estrogen pellets. On the other hand, we did not observe as dramatic an improvement in clinical phenotype as seen during pregnancy by estrogen supplementation.
The pathological hallmark of GLD is demyelination with apoptotic loss of the oligodendrocytes and infiltration of PAS-positive multinucleated peripheral macrophages (‘globoid cells’). Our findings suggest a possible anti-inflammatory effect of pregnancy. MCP-1 may mediate macrophage infiltration from the periphery to the CNS and produce further disease progression when overexpressed in the brain. Estrogen has been reported to decrease MCP-1 mRNA (24). Thus, the reduced expression of MCP-1 and TNF- in the brain of pregnant saposin A–/– mice may be an important factor to explain the improved phenotype.
In the peripheral organs, estrogen increases macrophage phagocytosis and has an anti-inflammatory effect (25,26). Estrogen receptors, ER and ERß, are expressed not only in neurons but also in cells of glial linage and microglia (12,13,27). Increasing evidence suggests that estrogen has anti-inflammatory effects on microglial activation (28–33). In view of the characteristic macrophage infiltration and reduced expression of some cytokines during pregnancy, we examined ER expression in the CNS of saposin A–/– mice. Therefore, it is conceivable that the high level of estrogen during pregnancy may act on the infiltrating macrophages and microglia in GLD, which intensely express ERs, and alleviate the disease by altering the expression of cytokines and consequently reducing the secondary inflammatory process. Our finding that E2 administration could substantially duplicate the protective effect of pregnancy supports this hypothesis.
Several reports suggested the protective effect of pregnancy in inflammatory demyelinating diseases. Women with multiple sclerosis (MS) may experience clinical remissions during pregnancy and exacerbations postpartum (34). Pregnant animals develop less severe experimental autoimmune encephalomyelitis (EAE) and estrogen administration can suppress EAE (15,16,35). ER has been demonstrated on T-cell subtypes and complete protection against EAE by a combined T-cell receptor plus estrogen therapy has been reported (15). Although many other complicating factors must be considered, the increased level of sex hormones during pregnancy is likely to be important. MS and EAE are peripheral T-cell-mediated inflammatory demyelinating diseases (15), whereas GLD is a demyelinating disease caused by genetically determined GALC deficiency with characteristic infiltration of PAS-positive peripheral macrophages (1). In contrast to MS or EAE, T cells may not play a predominant role in GLD, because there is no significant inflammatory T-cell infiltration in GLD (4). The role of ER on the macrophages and activated glial linage cells in GLD and the precise mechanism of E2 action remain to be established. Studies using ER antagonists or crossbreeding experiments between ER–/– and saposin A–/– mice should facilitate this investigation. On the other hand, it is also important to keep in mind that sex hormones may play an important role in maintenance and repair of the myelin sheath (36).
Although E2 could be highly effective in reducing neuropathology in saposin A–/– mice, the treatment was not without complications. It had adverse effects on the reproductive tract (overt enlargement of the uterus in females and shrinkage of the testis in males). We also found evidence of urinary obstruction (17). Furthermore, the timing of the treatment and the dosage must be critically important. We tested E2 pellet implantation in twitcher mice at 30 days, when the disease is already fully developed, but saw no effect (data not shown). Thus, the protective effect of E2 might be expected only when it is administered before active disease process begins (16). It would be of interest to implant the E2 pellet to twitcher mice, for example, at 10 days. We have examined only 17ß-estradiol as the most obvious candidate. Pregnancy affects the entire body metabolism in a complex manner and it is almost certainly simplistic to single out only 17ß-estradiol as the effector of the observed protective effects (33,37). More detailed follow-up investigations are warranted.
There is yet no truly effective treatment for most genetic leukodystrophies. Although not without side effects, hormonal administration might be worth a consideration as a possible supplementary treatment for GLD, and possibly for other genetic leukodystrophies, such as metachromatic leukodystrophy or adrenoleukodystrophy.
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MATERIALS AND METHODS |
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Animal care
All animal protocols used in these studies have been approved by the Internal Review Board of our university. Mice were maintained with access to water ad libitum. All mice were closely observed throughout their lives. Body weight was recorded daily as an objective parameter for development and progression of the disease. In order to determine the natural course of the disease, some mice were allowed to live as long as they could be maintained humanely according to the acceptable practice of laboratory animal care but without forced feeding or other extraneous interventions. For the series of experiments to see the effect of pregnancy in the saposin A–/– mice, we used only offspring of saposin A–/– mating. All pups were genotyped soon after birth and then again randomly spot-checked when they were killed for tissue sampling. This design excludes any possible influence during pregnancy by normal saposin A carried by fetuses, since all fetuses are saposin A–/–. We compared the clinical, pathological, biochemical and immunological phenotype among male, virgin female (never mated) and pregnant female sapsosin A–/– mice (kept mating with male saposin A–/– mice after weaning). All pregnant female saposin A–/– mice were pregnant at the time of sampling for the pathological, biochemical and immunological evaluation. All mice were F2 generation and the genetic background was still a mixture of 129 SV and C57BL6/J.
Genotyping of saposin A–/– mice, quantitation of brain psychosine and histopathology
These were done as previously described by Matsuda et al. (3).
Immunohistochemistry of estrogen receptors
After fixation, the brain and spinal cord were dissected, immersed in 20% sucrose for cryoprotection overnight and frozen with isopentane in Histobath (Shandon, Pittsburgh, PA). For sectioning, the samples were cut at 7 µm thickness with a cryostat and thaw-mounted on a glass slide. The mounted sections were air-dried for 30 min. Sections were rinsed with phosphate-buffered saline (PBS) and endogenous peroxidase was blocked by incubation with 0.3% hydrogen peroxide in distilled water for 20 min at room temperature. They were then washed with PBS and endogenous avidin/biotin binding was blocked with avidin D, followed by biotin (Vectastatin, Vector Laboratories, Burlingame, CA). After washing by PBS, the sections were incubated with 1.5% normal rabbit serum for Mac1 and with goat serum for ERs (ER and ERß) in PBS for 1 h at room temperature. The sections were then incubated overnight with each primary antibody, the anti-Mac1 antibody (rat IgG) (Roche Diagnostics, Indianapolis, IN) diluted 1:100 in PBS, anti-ER antibody (MC-20) (Santa Cruz Biotechnology, Santa Cruz, CA) (rabbit IgG) diluted 1:250 in PBS and anti-ERß antibody (H-150) (Santa Cruz Biotechnology) (rabbit IgG) diluted 1:250 in PBS at room temperature. The next day, the sections were washed in PBS containing 0.01% Triton X-100 and incubated with secondary biotinylated anti-rat IgG for Mac1 or anti-rabbit IgG for ERs (Vector Laboratories) diluted 1:200 in PBS for 1 h at room temperature and washed in PBS containing 0.01% Triton X-100, incubated with ABC solution (Elite ABC kit; Vector Laboratories, Palo Alto, CA). Immunoreactions were visualized by diaminobenzidine. In order to control for non-specific staining, additional sections were subjected to the immunostaining procedure without primary antibody. No staining was observed in control sections. The experiments were repeated three times using three different mice sections.
Expression of immune-related molecules in the brain of saposin A–/– mice by semiquantitative RT–PCR
The differential expression of variable cytokines and a TNF- receptor in the brain of saposin A–/– mice was analyzed by RT–PCR. Whole brain tissues from saposin A–/– mice (male, virgin female and pregnant female) and wild-type mice (male and virgin female) were collected. Mice were killed by decapitation and the brains were rapidly dissected and total RNA was isolated from each whole brain using TRI reagent (Sigma, St. Louis, MO) following the manufacturer’s protocol. Total RNA (2.5 µg) was reverse-transcribed using the SuperScript first-strand cDNA synthesis system (Gibco BRL, Gaithersburg, MD) and oligo (dT)12–18 primers. The cDNA was then amplified using specific primers for mouse IL-10, MCP-1, TNF-, TNFR2 (p75) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. The primers used were: sense, 5'-TCCTTAATGCAGGACTTTAAGGGTTACTTG-3' and antisense, 5'-GACACCTTGGTCTTGGAGCTTATTAAAATC-3' for mouse IL-10 (240bp); sense, 5'-CTCACCTGCTGCTACTCATTC-3' and antisense, 5'-GCATGAGGTGGTTGTGAAAAA-3' for mouse MCP-1 (350 bp); sense, 5'-TCTCATCAGTTCTATGGCCC-3' and antisense, 5'-GGGAGTGAGCAAGGTACAAC-3' for mouse TNF- (212 bp); sense, 5'-GCCCAGCCAAACTCCAAGCAT-3' and antisense, 5'-TGGAACTGGGTGCTGTGGTCA-3' for mouse TNFR2 (281 bp); sense, 5'-CCATGGAGAAGGCCGGGG-3' and antisense, 5'-CAAAGTTGTCATGGATGACC-3' for mouse GAPDH (194 bp) (11). The optimized numbers of PCR cycles allowing the signal to be in the linear portion of the amplification curve were 35 cycles for IL-10, 35 cycles for MCP-1, 30 cycles for TNF-, 30 cycles for TNFR2 and 17 cycles for GAPDH. A negative control lacking template cDNA was included in each RT–PCR. For quantitative analysis, the stained gel was scanned with an image scanner (ScanJet ADF; Hewlett-Packard) and the bands’ density was digitized using the automated digitizing software (UN-SCAN-IT gel Version 5.1; Silk Scientific Inc., Orem, UT). The values were normalized for the GAPDH bands.
Estrogen implantation experiment
The 3 mm 60 day time-release pellets containing 15 mg of 17ß-estradiol (E2), in a cholesterol matrix (Innovative Research of America, Sarasota, FL) were subcutaneously implanted to 30-day-old virgin female (n = 4) and male (n = 4) saposin A–/– mice. The 15 mg E2 pellets maintain the high pregnancy levels (5000–10 000 pg/ml) of estrogen over a period of 60 days (15–17). Control saposin A–/– mice (two females and two males) were sham-operated but received no estrogen pellet. In the natural course of the disease, saposin A–/– mice at 30 days are indistinguishable from wild-type mice and the GLD pathology is rarely detected and, at 90 days, the pathology is fully developed. For pathological evaluation, mice were killed after the 60 day estrogen treatment at around 90 days of age. The preservation of myelin and the degree of infiltration of the PAS-positive macrophages were blindly evaluated by two neuropathologists.
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ACKNOWLEDGEMENTS |
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Ms Elise Cash, Rachel Selinger, Clarita Langaman and Mr Joe Langaman provided technical assistance in tissue preparation and cell culture. This work was supported in part by research grants from the USPHS, RO1-NS24289, RO1-NS24453 and Mental Retardation Research Center Core Grant, P30-HD03110, and a research grant from the European Leukodystrophy Association (ELA) to M.T.V.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Neuroscience Center, CB7250, University of North Carolina, Chapel Hill, NC 27599, USA. Tel: +1 919 966 2405; Fax: +1 919 966 1844; Email: kuni.suzuki@attglobal.netPresent address:Dr Yuko Saito, Department of Neuropathology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan
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REFERENCES |
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