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Temporal and Spatial Expression of Murine Acid β-Glucosidase mRNA
Molecular Genetics and Metabolism 74, 426 – 434 (2001) doi:10.1006/mgme.2001.3258, available online at http://www.idealibrary.com on
Temporal and Spatial Expression of Murine Acid -Glucosidase mRNA Elvira Ponce,* David P. Witte,† Alida Hung,‡ and Gregory A. Grabowski* ,1 *Division of Human Genetics and †Division of Pathology, Children’s Hospital Medical Center, Cincinnati, Ohio; and ‡Escuela de Medicina J.M. Vargas UCV, Caracas, Venezuela Received August 28, 2001, and in revised form September 14, 2001; published online November 30, 2001
cosylceramides; GC) in mammals. This enzyme’s activity is present in lysosomes of all nucleated cells and yields glucose and ceramide. In Gaucher disease (GD), an autosomal inherited disorder, defective GCase function results in progressive lysosomal accumulation of GC and subsequent multiorgan dysfunction (1). Ablation of the mouse GCase gene (gba) results in undetectable enzyme activity (2), and a neonatal lethal phenotype similar to that in some severe, early onset GC in humans (3). Despite GCase’s ubiquitous presence, storage of glucocerebrosides occurs predominantly in cells of monocyte/ macrophage lineage of the liver, spleen, lymph nodes, and bone marrow (1). Three human GD clinical variants, Types 1, 2, and 3, differ in age of onset and CNS involvement. GD Type 1 is nonneuronopathic and is the most common variant. Hepatosplenomegaly, hypersplenism, and bony disease are hallmarks. Bony manifestations include osteopenia, osteonecrosis, and failed remodeling (4). The phenotypes range from mild to very severe with age of onset of signs from ⬍1 to ⬎80 years (1). The neuronopathic variants (Type 2, acute, or Type 3, subacute) present in the first months to 1 year, respectively. Hepatosplenomegaly and brainstem abnormalities develop early in Type 2, and variable visceral and CNS progressive involvement are present in Type 3 disease. Types 2 and 3 represent a spectrum of variable CNS disease occurring from the newborn period into the second decade (1). In the CNS a rostral to caudal gradient of accumulated GC has been found in patients with neuronopathic variants of GD (5), suggesting tissue-specific requirements of GC or GCase. The tissue-specific involve-
The hydrolysis of glucosylceramide (GC) to ceramide and glucose requires the action of the lysosomal enzyme, acid -glucosidase (GCase), encoded by gba in the mouse. Gaucher disease, an autosomal recessive disorder, results from the inherited deficiency of this enzyme. Although enzyme activity is present in all mammalian tissues, the patterns of mRNA expression have not been explored. In situ hybridization analyses of mouse embryonic, newborn, and adult tissues were conducted to evaluate the spectrum of gba mRNA expression. Signals were present in all tissues and cell types. Distinct patterns of differential expression were identified in specific tissues and cell types, and at defined developmental stages. Differential expression was first observed around E14 in the intestinal tract, kidneys, skeletal system, and skin. At E18, moderate intensity signals were in adipocytes of brown fat and pancreatic cells. Differential expression remained in skin, bone, and the GI tract postnatally. In the postnatal and adult animals increasing expression was observed throughout the CNS, esophageal epithelium, intestinal villi, pancreas, and thymus and lymph node capsular cells. These tissue-, cell-, and developmental stage-specific variations of the gba mRNA level indicate major developmentally regulated changes in the expression pattern of gba in the late gestational period and postnatally. © 2001 Elsevier Science
Acid -glucosidase (GCase; EC 3.2.1.45) cleaves the -glucosidic linkage of glucocerebrosides (glu1
To whom correspondence should be addressed at Children’s Hospital Research Foundation, Division of Human Genetics, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. Fax: (513) 636-2261. E-mail: [email protected]. 426 1096-7192/01 $35.00 © 2001 Elsevier Science All rights reserved.
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ment or severity in GD disease is not well understood and correlates to a limited extent with genotype. The locus maps to human chromosome1q21 (GBA) and the syntenic mouse chromosome region 3 (E3– F1) (gba) (6). Thrombospondin (TSP3), episialin (MUC1), metaxin (MTX) and pseudogenes for GBA(⌿GBA) and MTX (⌿MTX) are components of the human region containing GBA. The chromosomal arrangement is GBA, ⌿MTX, ⌿GBA, MTX, and TSP3 and spans 45 kb. The GBA and metaxin pseudogenes also are present in rhesus monkeys, but are absent in rodents and other species, indicating recent duplication of these genes (7). Transcription of the ⌿GBA occurs, but the product is not translated into a functional protein (8). Characterization of the human GBA promoter revealed 2 TATA boxes and two possible CAAT boxes but no SP1-binding sites. A position-independent enhancer is located within exon 1 (9,10). Transfection reporter (CAT) experiments using 650 bp of 5⬘-flanking sequence suggested that the GBA promoter has tissue/cellular, rather than a housekeeping, specificity. Northern blots with mRNA’s from Hela cells, and normal and GD individuals, show major mRNA species of 2.5 kb, and two other minor mRNAs, 5.6 and ⬃2.0 kb, originate from incomplete splicing of nuclear transcripts or alternative transcription initiation and/or polyadenylation (11). Macrophages and B-cells exhibit very low levels of GBA mRNA while skin fibroblasts and promyelocytes have intermediate levels, and epithelial cells have high levels. To gain insight into the tissue-specific pathology and pathogenesis of GD, the mouse gba mRNA expression patterns were evaluated in prenatal, newborn (2 and 4 days old), and adult tissues by in situ hybridization. The results show temporal and developmental control of the gba expression at the transcriptional level. MATERIALS AND METHODS Northern blot analysis. A 768-bp 32P-labeled mouse GCase cDNA (EcoRI 102 nt upstream of the ATG to EcoRV in Exon 6) was hybridized to Swiss Webster and BALB C mouse poly(A) ⫹ RNA (2 g/ lane) from whole embryos and a variety of adult tissues. Hybridization with a human -actin cDNA probe was used for RNA quality control. Quantitation of bound probe signals was by phosphoimager scanning of 24-h exposed screens.
In situ hybridization. Mouse gba 35S-labeled sense and antisense riboprobes were synthesized by in vitro transcription (12) from linearized templates containing the same fragment as for Northern blots. In situ hybridizations of B6C3F1/J (C57BL/6J ⫻ C3H/ HeJ) mouse tissues (Harland Animal Inc., Indianapolis, IN) were performed as described (12). Briefly, cryosections of 4% paraformaldehyde-fixed and embedded tissues were postfixed and prepared for in situ hybridization. The sections were hybridized with 35S-UTP-labeled ⬃1-kb sense or antisense gba riboprobes under high stringency conditions including 50% formamide. Hybridization was followed by ribonuclease A/T1 digestion and washed under progressively increasing stringency conditions, including 0.1X SSC at 55°C. This was followed by dehydration in graded ethanol solutions, dipping in Kodak NTB2 emulsion, and exposure at 4°C for 10 –15 days. Slides were developed and stained with hematoxylin and eosin (HE). Positive signals, obtained with the antisense probe, appear as white or light pink grains under dark-field microscopy. Duplicate sections hybridized with the sense probe were used as negative controls. RESULTS Northern Blots A major gba transcript (2.5 kb) was present in mRNA blots from whole embryos at Days E7, 11, 15, and 17 and adult mouse tissues. Minor bands of ⬃5.2 and ⬃2.1 kb were also detected (not shown). The ratios of mRNA forms did not vary during development. Prenatally, Northern analysis revealed the highest level mRNA expression at E7 (assigned, 100%). The lowest prenatal gba mRNA levels were detected at E11 (13%). These increased toward the end of gestation (E15, 40%, and E17, 30%). Similarly analyzed adult mouse tissues showed that gba mRNA levels were highest in the liver (assigned, 100%); intermediate in kidney (36%) and heart (21%); and low in lung (14%), brain (13%), and testis (12%). The lowest gba mRNA levels were in the spleen (7%) and skeletal muscle (7%). In Situ Hybridization Prenatal studies. Embryos were examined by in situ hybridization for expression of GCase mRNA from E6.0 to E18. There was no specificity to the expression pattern as signal was present uniformly throughout the embryonic cells. In general, until
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TABLE 1 Relative GCase mRNA Signal Intensity in Prenatal Tissues by in Situ Hybridization Stage E5.5 E10
Tissue
mRNA
Cell type
Developing trophoblast Maternal adjacent tissues Primitive mesenchime Neural tissues
⫹ ⫹⫹ ⫹ ⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹
Cells of trophoectoderm, endoderm, and embryonic ectoderm Decidual and sinusoidal cells All cell types Most cell types Cerebral ventricular epithelial lining Auditory vesicle cochlear epithelial lining Lens epithelial layer All retinal layer cells Most cell types Most cell types Cerebral ventricular choroid plexus Pituitary and cranial nerve V cells Upper neural tube and ventral spinal cord cells Medulla Pigmented and sensory layer cells Lens epithelial cells All cell types Yolk sac Most cell types Similar to the E12–13 stage Spinal dorsal root ganglion cells Spinal cord meningeal cells and ventral horn cells Otic capsule and cochlear epithelial cells Ganglion cell layer Cells of the lens and retinal layers Developing epidermis outer epithelial cell layers Chondrocytes Epithelial cells Intestinal villi epithelial cells Metanephros tubular epithelial cells Most cell types. Expression patterns similar to E14–16 Adipocytes Cardiocytes Myocytes Acinar glands and islet epithelial cells
Ear Eye E12–13
Overall embryo Neural tissues
Eye
E14–16
Liver Placenta Overall embryo Neural tissues
Ear Eye
E18
Skin Bone Salivary glands Intestine Kidney Overall embryo Brown fat Heart Skeletal muscle Pancreas
⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹
Note. The mRNA signal intensity was scored as follows: ⫹, very low; ⫹⫹, low; ⫹⫹⫹, moderate; ⫹⫹⫹⫹, high.
E14, the expression level of gba was very low with signal that was detectable just above background levels. The CNS showed some differential expression in specific regions (Table 1). By E14 the expression pattern showed the first signs of differentiation with stronger signal evident in the epithelial cells lining the small bowel intestinal villi and the large intestine. Similarly weak expression was observed for the first time in the ribs, tubular epithelium of the developing kidneys, and the epidermal layer of the developing skin. The mucosal epithelium lining the forestomach also showed slightly increased expression. In the E16 –E18 embryos the expression patterns in these structures became slightly more intense with increasing gestational age. By E18 the
epithelial cells lining the intestinal villi showed increasing, but still weak, signal as did the epidermis and in the conversional zones of the developing bones. The brain at this stage showed low to moderate expression with little distinctive patterns to the expression. In the extraembryonic tissue the trophoblastic cells in the E8 placenta showed weak signal and the yolk sac epithelium showed weak to moderate staining in the developing placenta. Postnatal studies (Table 2, Fig. 1). Differential expression patterns of GCase mRNA were clearly evident in the 2-day-old newborns (Figs. 1A–K) and in 4-week-old mature mouse tissues (Figs. 1L–P). In particular, in the CNS expression became increas-
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TABLE 2 Relative GCase mRNA Signal Intensity in Postnatal Tissues by in Situ Hybridization Stage Days 2–4
Tissue Overall expression Neural tissues
Eye Skin Bone Lungs Esophagus Forestomach Intestine Pancreas Liver Kidney Testis Thymus Intestinal lymph node Adult
Overall expression Neural tissues Skin Esophagus Intestine Pancreas Kidney Adrenals Testis Lymph node Lungs
mRNA
Cell type
⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹
Most cell types Purkinje cell layer Cerebral, cerebellar neurons and meningeal cells Glial cells Spinal cord and spinal dorsal root ganglion cells Ganglion, and rods and cones layers Other retinal cells Mature epithelial cells Hair follicle and sweat gland cells Conversional zone cells All cell types Squamous epithelial cells Epithelial cells Mucosal epithelial cells Serosal cells Acinar glands and islet epithelial cells All cell types Collecting tubular epithelial cells Other nephron epithelial cells Epithelial cells lining developing seminiferous tubules Thymocytes Capsular lining cells Other cell types Most tissues Cerebral hippocampus and thalamic nuclei neurons Cerebellar Purkinje cells Mature epithelial epidermal layer Hair follicle and sweat glands cells Squamous epithelial cells Mucosal and serosal cells Acinar glands and islets Collecting tubular epithelial, juxtamedullary, and glomerular cells Most cells Sertoli cells Vas deferens epithelium Capsular lining cells All cell types
Note. The mRNA signal intensity was scored as follows: ⫹, very low; ⫹⫹, low; ⫹⫹⫹, moderate; ⫹⫹⫹⫹, high.
ingly intense in all the neuronal cells compared to the embryonic brain stages where the signal was diffuse but weak. All the neuronal layers showed strong expression in the 2-day and 4-week-old postnatal mice. The more densely populated neuronal layers such as the hippocampus showed bright bands of signal in contrast to adjacent layers mostly populated by glial cell populations (Figs. 1I–L). Neuronal cells in the spinal cord and dorsal root ganglia showed similar strong expression in the postnatal mice. In addition to the neuronal cells in the CNS, strong expression was also detected in the meningeal cells (Figs. 1I and 1K) and epithelial cells of the
choroid plexus (data not shown). In the postnatal eye the retina showed strong signal in the ganglion cell layer as well as in the inner and outer nuclear layers. Differential expression was also observed in postnatal non-CNS tissues. In the skin GCase mRNA expression continued to be observed in the outer epidermal cell layers at Neonatal Day 2 and at 4 weeks (Figs. 1A and 1M, respectively). Strong signal also was observed in hair follicles (Fig. 1M). In the lymph nodes, the sinusoidal capsule showed signals of moderate intensity (Fig. 1O) with no significant signal in the lymphoid cell populations. The spleen
FIG. 1. In situ hybridization profiles of postnatal tissues using gba mRNA antisense probes. Various tissues from Neonatal Days 2– 4 (A–K) and 4-week old adult mice (L–P). (A–D) Postnatal Day 2 tissues: (A) Skin, outermost epidermal layers (arrowhead); (B) neonatal forestomach (squamous cell mucosa, arrowhead); (C) small intestine; cross section of villi (arrowhead) and serosal cell layers (arrow). (D) Bone: Signals of moderate intensity in osteoblasts (arrowhead) of bony trabeculae at the conversional zone; (E–H) Postnatal Day 4; (E) a negative control using the sense gba RNA. (F and G) Highest intensity signals throughout the mucosal epithelial cells of the small intestine (arrowheads) with moderate signals at the serosal surface. The asterisks are smooth muscle cells (negative). (H) Visceral organs:
MURINE GCase mRNA EXPRESSION
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FIG. 1—Continued
The pancreas (p), kidney tubular epithelial cells (kd), and epithelial cells lining the developing seminiferous tubules (t) and tubules of the epididymus (e) had high to moderate signals, skeletal muscle fibers (skm) show weak signal. (I–L) Various CNS regions from 2- and 4-day-old brains: (I) Cerebral cortex (layers III and V, arrowheads); (J) cerebellar cortex (Purkinje cell layer, arrowheads); (K) cerebral cortex (h, hippocampus; meninges arrowhead); (L) hippocampus (h) of a 4-week-old adult. (M–P) adult tissues: (M) skin with outer epidermal cells (arrow) and hair follicles (arrowhead); (N) squamous epithelium of the esophagus (arrowheads); smooth muscle cells (arrow) were negative; (O) lymph node (arrowhead-capsular lining cells); (P) testes (Sertoli cells; arrowhead). (All photomicrographs dark-field illumination, Panels A, C, E–G, I–L, N–P, ⫻100; Panels B, D, H, and M, ⫻40; Panel G, ⫻200).
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also showed very low expression. Expression in the bone was most intense in the conversional zones in areas of new bone growth (Fig. 1D). In contrast, little expression was observed in skeletal muscle tissue or cardiac muscle cells. Some of the most intense expression was observed in the digestive system. The squamous epithelium lining the esophagus and forestomach (Figs. 1B and 1N) showed strong signal. The epithelial cells lining the small intestine also showed strong expression (Figs. 1C, 1F, and 1G) and the colonic glandular epithelium showed moderate expression levels. The pancreas also was positive (Fig. 1H) and the liver showed only weak expression. In the genitourinary tract expression was detected in the 2-day-old newborn at weak levels in the tubular epithelium. Expression in the 4-day-old newborn kidney also was diffuse in the tubular epithelium, but slightly more intense. Expression was also observed in the more immature tubules and glomeruli in the nephrogenic zone just under the capsule (Fig. 1H). In the testis (Fig. 1P) signal was detected in the Sertoli cells lining the seminiferous tubules as well as in the epithelial cells lining the epididymus and vas deferens. DISCUSSION In situ hybridization using gba antisense RNA showed generalized low level expression early in gestation with gradual appearance of differential expression appearing around gestational age E14 and significantly increasing at term and into adulthood. During early embryonic development, except for trophoblastic cells, gba mRNA was at relatively low levels and high levels were not present in cells or regions that had high cellular turnover and/or remodeling. With increased apoptosis or remodeling during development a regional increase in gba mRNA might have been expected because of the accompanying increased metabolic demands due to membrane turnover. In the postnatal brain and mature brain increasing expression was observed in all neuronal populations throughout the brain. In contrast to the bands of strong signal observed in the neuronal layers such as the hippocampus, cortical layers, and cerebellar layers, the adjacent myelinated or myelinating regions of the CNS did not show significant levels of gba mRNA expression as had been observed with aspartylglucosaminidase (13). All variants of Gaucher disease have CNS pathology that includes periadventitial accumulations of Gaucher cells in the Virchow-Robinow spaces, and
around venules of the cerebral and cerebellar cortical regions (14,15). These cells are extraneural and probably derive from the monocyte/macrophage system secondary to the storage of GC derived from visceral sources as determined by fatty acid composition analyses (16). These cells also accumulate in a rostral to caudal gradient (17) and probably account for the majority of the similar concentration gradient of GC accumulation in brains of Type 2 and 3 patients. To understand the potential relationship between GCase expression and the pathology of Gaucher disease two different mechanisms must be considered. In the visceral tissues, the primary pathophysiology relates to the macrophage and its processing of engulfed cellular debris and old and dying cells. Such debris is derived primarily from the blood-formed elements that are phagocytosed and is present by macrophages within the liver, spleen, and bone marrow. Accumulations of GC must result from phagocytosis and the inability of the residual enzyme activity in these macrophages to completely eliminate the presented GC from glucosphingolipid precursors. This is supported by the observation of Gaucher-like cells in non-Gaucher individuals who have chronic myelogenesis leukemia. The large amounts of GC presented to macrophages is from the massive proliferation of myelocytic cells whose glucosphingolipids require processing through the sequential hydrolytic system (18,19). The mass of this substrate presentation overwhelms the degradative capacity of even normal amounts of enzyme and Gaucher-like cells accumulate. It might be anticipated that if larger amounts of functional GCase were present in these normal cells that such cells would not accumulate GC. Thus, some imbalance of substrate presentation (glucosylceramide flux) and GCase amount in cells is essential for substrate accumulation and Gaucher cell development in visceral tissues. Interestingly, higher levels of gba mRNA expression were found in a variety of epithelial cells in visceral organs, including the skin. These epithelial cells are desquamated and are eliminated from the body. Thus, they do not have the chance to accumulate substrate, even if it could be phagocytosed and exceeded the capacity of the residual activity to catabolize this material. Similarly, the nonphagocytic cells of the liver and lymphoid system do not accumulate significant amounts of GC in Gaucher disease since the normal amount of presented GC likely derives from endogenous cellular synthesis and does not exceed the degradative capacity of those cells.
MURINE GCase mRNA EXPRESSION
The pathophysiology in the brain is different since neuronal involvement and neuronal death are major events that lead to degeneration. Excluding the accumulations of macrophages (peripherally derived) in the Virchow-Robinow spaces, a gradient of involvement of neurons from rostral to caudal is not apparent in the brains of affected Type 2 patients (5,17). However, regional variation of neuronal cell loss does occur. This includes more distinct involvement of layers III and V in the cerebral cortex, hippocampal pyramidal cells, neurons of the thalamic and basal ganglia, and the dentate nucleus of the cerebellum. These areas show the highest degrees of neuronal cell loss with subsequent gliosis. Based on the mRNA expression, gba appears uniformly throughout the CNS. Regional variation may relate to species differences between humans, where the pathology has been described and the expression pattern described in the mouse brain. Alternatively the regional differences described in the pathology of human gba deficiency may be the result of multiple factors such as differences in metabolic activity, vascular perfusion, or other contributing factors which may make certain neuronal populations more susceptible to degeneration as a result of gba deficiency. Previously, glucosphingosine, the deacylated analogue of GC, was suggested, in analogy to galactosphingosine in Krabbe disease, as the toxic compound leading to neuronal death (20). Although this remains a viable pathophysiologic explanation, direct proof is lacking. The toxicity of accumulated GC has not been excluded and, indeed, the inflammatory reaction surrounding some of the accumulated macrophages in the Virchow-Robinow spaces (21) and the overexpression of macrophage genes in Gaucher cells (22) suggest the possibility of direct involvement and/or toxicity of GC in the pathophysiologic process of the central nervous system and visceral involvement in neuronopathic Gaucher disease.
model of Gaucher’s disease from targeted disruption of the mouse glucocerebrosidase gene. Nature 357:407– 412, 1992. 3.
Sidransky E, Sherer DM, Ginns EI. Gaucher disease in the neonate: A distinct Gaucher phenotype is analogous to a mouse model created by targeted disruption of the glucocerebrosidase gene. Pediatr Res 32:494 – 498, 1992.
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Kaye EM, Ullman MD, Wilson ER, Barranger JA. Type 2 and type 3 Gaucher disease: A morphological and biochemical study. Ann Neurol 20:223–230, 1986.
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Bornstein P, McKinney CE, LaMarca ME, et al. Metaxin, a gene contiguous to both thrombospondin 3 and glucocerebrosidase, is required for embryonic development in the mouse: Implications for Gaucher disease. Proc Natl Acad Sci USA 92:4547– 4551, 1995.
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Zimran A, Sorge J, Gross E, Kubitz M, West C, Beutler EA. Glucocerebrosidase fusion gene in Gaucher disease. Implications for the molecular anatomy, pathogenesis, and diagnosis of this disorder. J Clin Invest 85:219 –222, 1990.
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Doll RF, Bruce A, Smith FI. Regulation of the human acid -glucosidase promoter in multiple cell types. Biochim Biophys Acta 1261:57– 67, 1995. Doll RF, Smith FI. Regulation of expression of the gene encoding human acid -glucosidase in different cell types. Gene 127:255–260, 1993. Graves PN, Grabowski GA, Ludman MD, Palese P, Smith FI. Human acid beta-glucosidase: Northern blot and S1 nuclease analysis of mRNA from HeLa cells and normal and Gaucher disease fibroblasts. Am J Hum Genet 39:763–774, 1986. Ponce E, Witte DP, Hirschhorn R, Huie M, Grabowski GA. Temporal and spatial expression of acid alpha-glucosidase mRNA in mice. Am J Pathol 154:1089 –1096, 1999. Uusitalo A, Tenhunen K, Heinonen O, et al. Toward understanding the neuronal pathogenesis of aspartylglucosaminuria: Expression of aspartylglucosaminidase in brain during development. Mol Genet Metabol 67:294 –307, 1999. Adachi M, Wallace BJ, Schneck L, Volk BW. Fine structure of central nervous system in early infantile Gaucher’s disease. Arch Pathol 83:513–526, 1967. Volk BW, Wallace BJ, Adachi M. Infantile Gaucher’s disease: Electron microscopic and histochemical studies of a cerebral biopsy. J Neuropathol Exp Neurol 26:176 –177, 1967. Nilsson O, Svennerholm L. Accumulation of glucosylceramide and glucosylsphingosine (psychosine) in cerebrum and cerebellum in infantile and juvenile Gaucher disease. J Neurochem 39:709 –718, 1982. Banker BQ, Miller JQ, Crocker AC. The cerebral pathology of infantile Gaucher’s disease. In Cerebral Sphingolipidoses (Aronson SM, Volk BM, Eds.). New York: Academic Press, pp 73–99, 1962. Albrecht M. “Gaucher cells” in chronic myelocytic leukemia. Klin Wochenschr 47:778 –784, 1969.
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ACKNOWLEDGMENT This work was supported by NIH Grant RO1 DK 36729 (G.A.G.).
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Dosik H, Rosner F, Sawitsky A. Acquired lipidosis: Gaucherlike cells and “blue cells” in chronic granulocytic leukemia. Semin Hematol 9:309 –316, 1972. 20. Suzuki K, Suzuki Y. Galactosylceramide lipidosis: Globoid cell leukodystrophy (Krabbe disease). In The Metabolic Basis of Inherited Disease (Scriver CR, Beaudet AL, Sly WS, Valle D, Eds). New York: McGraw-Hill, pp 1699 –1720, 1989.
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Conradi NG, Kalimo H, Sourander P. Reactions of vessel walls and brain parenchyma to the accumulation of Gaucher cells in the Norrbottnian type (type III) of Gaucher disease. Acta Neuropathol 75:385–390, 1988. 22. Moran MT, Schofield JP, Hayman AR, Shi GP, Young E, Cox T. Pathologic gene expression in Gaucher disease: Up-regulation of cysteine proteinases including osteoclastic cathepsin K. Blood 96:1969 –1978, 2000.
Temporal and Spatial Expression of Murine Acid -Glucosidase mRNA Elvira Ponce,* David P. Witte,† Alida Hung,‡ and Gregory A. Grabowski* ,1 *Division of Human Genetics and †Division of Pathology, Children’s Hospital Medical Center, Cincinnati, Ohio; and ‡Escuela de Medicina J.M. Vargas UCV, Caracas, Venezuela Received August 28, 2001, and in revised form September 14, 2001; published online November 30, 2001
cosylceramides; GC) in mammals. This enzyme’s activity is present in lysosomes of all nucleated cells and yields glucose and ceramide. In Gaucher disease (GD), an autosomal inherited disorder, defective GCase function results in progressive lysosomal accumulation of GC and subsequent multiorgan dysfunction (1). Ablation of the mouse GCase gene (gba) results in undetectable enzyme activity (2), and a neonatal lethal phenotype similar to that in some severe, early onset GC in humans (3). Despite GCase’s ubiquitous presence, storage of glucocerebrosides occurs predominantly in cells of monocyte/ macrophage lineage of the liver, spleen, lymph nodes, and bone marrow (1). Three human GD clinical variants, Types 1, 2, and 3, differ in age of onset and CNS involvement. GD Type 1 is nonneuronopathic and is the most common variant. Hepatosplenomegaly, hypersplenism, and bony disease are hallmarks. Bony manifestations include osteopenia, osteonecrosis, and failed remodeling (4). The phenotypes range from mild to very severe with age of onset of signs from ⬍1 to ⬎80 years (1). The neuronopathic variants (Type 2, acute, or Type 3, subacute) present in the first months to 1 year, respectively. Hepatosplenomegaly and brainstem abnormalities develop early in Type 2, and variable visceral and CNS progressive involvement are present in Type 3 disease. Types 2 and 3 represent a spectrum of variable CNS disease occurring from the newborn period into the second decade (1). In the CNS a rostral to caudal gradient of accumulated GC has been found in patients with neuronopathic variants of GD (5), suggesting tissue-specific requirements of GC or GCase. The tissue-specific involve-
The hydrolysis of glucosylceramide (GC) to ceramide and glucose requires the action of the lysosomal enzyme, acid -glucosidase (GCase), encoded by gba in the mouse. Gaucher disease, an autosomal recessive disorder, results from the inherited deficiency of this enzyme. Although enzyme activity is present in all mammalian tissues, the patterns of mRNA expression have not been explored. In situ hybridization analyses of mouse embryonic, newborn, and adult tissues were conducted to evaluate the spectrum of gba mRNA expression. Signals were present in all tissues and cell types. Distinct patterns of differential expression were identified in specific tissues and cell types, and at defined developmental stages. Differential expression was first observed around E14 in the intestinal tract, kidneys, skeletal system, and skin. At E18, moderate intensity signals were in adipocytes of brown fat and pancreatic cells. Differential expression remained in skin, bone, and the GI tract postnatally. In the postnatal and adult animals increasing expression was observed throughout the CNS, esophageal epithelium, intestinal villi, pancreas, and thymus and lymph node capsular cells. These tissue-, cell-, and developmental stage-specific variations of the gba mRNA level indicate major developmentally regulated changes in the expression pattern of gba in the late gestational period and postnatally. © 2001 Elsevier Science
Acid -glucosidase (GCase; EC 3.2.1.45) cleaves the -glucosidic linkage of glucocerebrosides (glu1
To whom correspondence should be addressed at Children’s Hospital Research Foundation, Division of Human Genetics, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. Fax: (513) 636-2261. E-mail: [email protected]. 426 1096-7192/01 $35.00 © 2001 Elsevier Science All rights reserved.
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ment or severity in GD disease is not well understood and correlates to a limited extent with genotype. The locus maps to human chromosome1q21 (GBA) and the syntenic mouse chromosome region 3 (E3– F1) (gba) (6). Thrombospondin (TSP3), episialin (MUC1), metaxin (MTX) and pseudogenes for GBA(⌿GBA) and MTX (⌿MTX) are components of the human region containing GBA. The chromosomal arrangement is GBA, ⌿MTX, ⌿GBA, MTX, and TSP3 and spans 45 kb. The GBA and metaxin pseudogenes also are present in rhesus monkeys, but are absent in rodents and other species, indicating recent duplication of these genes (7). Transcription of the ⌿GBA occurs, but the product is not translated into a functional protein (8). Characterization of the human GBA promoter revealed 2 TATA boxes and two possible CAAT boxes but no SP1-binding sites. A position-independent enhancer is located within exon 1 (9,10). Transfection reporter (CAT) experiments using 650 bp of 5⬘-flanking sequence suggested that the GBA promoter has tissue/cellular, rather than a housekeeping, specificity. Northern blots with mRNA’s from Hela cells, and normal and GD individuals, show major mRNA species of 2.5 kb, and two other minor mRNAs, 5.6 and ⬃2.0 kb, originate from incomplete splicing of nuclear transcripts or alternative transcription initiation and/or polyadenylation (11). Macrophages and B-cells exhibit very low levels of GBA mRNA while skin fibroblasts and promyelocytes have intermediate levels, and epithelial cells have high levels. To gain insight into the tissue-specific pathology and pathogenesis of GD, the mouse gba mRNA expression patterns were evaluated in prenatal, newborn (2 and 4 days old), and adult tissues by in situ hybridization. The results show temporal and developmental control of the gba expression at the transcriptional level. MATERIALS AND METHODS Northern blot analysis. A 768-bp 32P-labeled mouse GCase cDNA (EcoRI 102 nt upstream of the ATG to EcoRV in Exon 6) was hybridized to Swiss Webster and BALB C mouse poly(A) ⫹ RNA (2 g/ lane) from whole embryos and a variety of adult tissues. Hybridization with a human -actin cDNA probe was used for RNA quality control. Quantitation of bound probe signals was by phosphoimager scanning of 24-h exposed screens.
In situ hybridization. Mouse gba 35S-labeled sense and antisense riboprobes were synthesized by in vitro transcription (12) from linearized templates containing the same fragment as for Northern blots. In situ hybridizations of B6C3F1/J (C57BL/6J ⫻ C3H/ HeJ) mouse tissues (Harland Animal Inc., Indianapolis, IN) were performed as described (12). Briefly, cryosections of 4% paraformaldehyde-fixed and embedded tissues were postfixed and prepared for in situ hybridization. The sections were hybridized with 35S-UTP-labeled ⬃1-kb sense or antisense gba riboprobes under high stringency conditions including 50% formamide. Hybridization was followed by ribonuclease A/T1 digestion and washed under progressively increasing stringency conditions, including 0.1X SSC at 55°C. This was followed by dehydration in graded ethanol solutions, dipping in Kodak NTB2 emulsion, and exposure at 4°C for 10 –15 days. Slides were developed and stained with hematoxylin and eosin (HE). Positive signals, obtained with the antisense probe, appear as white or light pink grains under dark-field microscopy. Duplicate sections hybridized with the sense probe were used as negative controls. RESULTS Northern Blots A major gba transcript (2.5 kb) was present in mRNA blots from whole embryos at Days E7, 11, 15, and 17 and adult mouse tissues. Minor bands of ⬃5.2 and ⬃2.1 kb were also detected (not shown). The ratios of mRNA forms did not vary during development. Prenatally, Northern analysis revealed the highest level mRNA expression at E7 (assigned, 100%). The lowest prenatal gba mRNA levels were detected at E11 (13%). These increased toward the end of gestation (E15, 40%, and E17, 30%). Similarly analyzed adult mouse tissues showed that gba mRNA levels were highest in the liver (assigned, 100%); intermediate in kidney (36%) and heart (21%); and low in lung (14%), brain (13%), and testis (12%). The lowest gba mRNA levels were in the spleen (7%) and skeletal muscle (7%). In Situ Hybridization Prenatal studies. Embryos were examined by in situ hybridization for expression of GCase mRNA from E6.0 to E18. There was no specificity to the expression pattern as signal was present uniformly throughout the embryonic cells. In general, until
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TABLE 1 Relative GCase mRNA Signal Intensity in Prenatal Tissues by in Situ Hybridization Stage E5.5 E10
Tissue
mRNA
Cell type
Developing trophoblast Maternal adjacent tissues Primitive mesenchime Neural tissues
⫹ ⫹⫹ ⫹ ⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹
Cells of trophoectoderm, endoderm, and embryonic ectoderm Decidual and sinusoidal cells All cell types Most cell types Cerebral ventricular epithelial lining Auditory vesicle cochlear epithelial lining Lens epithelial layer All retinal layer cells Most cell types Most cell types Cerebral ventricular choroid plexus Pituitary and cranial nerve V cells Upper neural tube and ventral spinal cord cells Medulla Pigmented and sensory layer cells Lens epithelial cells All cell types Yolk sac Most cell types Similar to the E12–13 stage Spinal dorsal root ganglion cells Spinal cord meningeal cells and ventral horn cells Otic capsule and cochlear epithelial cells Ganglion cell layer Cells of the lens and retinal layers Developing epidermis outer epithelial cell layers Chondrocytes Epithelial cells Intestinal villi epithelial cells Metanephros tubular epithelial cells Most cell types. Expression patterns similar to E14–16 Adipocytes Cardiocytes Myocytes Acinar glands and islet epithelial cells
Ear Eye E12–13
Overall embryo Neural tissues
Eye
E14–16
Liver Placenta Overall embryo Neural tissues
Ear Eye
E18
Skin Bone Salivary glands Intestine Kidney Overall embryo Brown fat Heart Skeletal muscle Pancreas
⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹
Note. The mRNA signal intensity was scored as follows: ⫹, very low; ⫹⫹, low; ⫹⫹⫹, moderate; ⫹⫹⫹⫹, high.
E14, the expression level of gba was very low with signal that was detectable just above background levels. The CNS showed some differential expression in specific regions (Table 1). By E14 the expression pattern showed the first signs of differentiation with stronger signal evident in the epithelial cells lining the small bowel intestinal villi and the large intestine. Similarly weak expression was observed for the first time in the ribs, tubular epithelium of the developing kidneys, and the epidermal layer of the developing skin. The mucosal epithelium lining the forestomach also showed slightly increased expression. In the E16 –E18 embryos the expression patterns in these structures became slightly more intense with increasing gestational age. By E18 the
epithelial cells lining the intestinal villi showed increasing, but still weak, signal as did the epidermis and in the conversional zones of the developing bones. The brain at this stage showed low to moderate expression with little distinctive patterns to the expression. In the extraembryonic tissue the trophoblastic cells in the E8 placenta showed weak signal and the yolk sac epithelium showed weak to moderate staining in the developing placenta. Postnatal studies (Table 2, Fig. 1). Differential expression patterns of GCase mRNA were clearly evident in the 2-day-old newborns (Figs. 1A–K) and in 4-week-old mature mouse tissues (Figs. 1L–P). In particular, in the CNS expression became increas-
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TABLE 2 Relative GCase mRNA Signal Intensity in Postnatal Tissues by in Situ Hybridization Stage Days 2–4
Tissue Overall expression Neural tissues
Eye Skin Bone Lungs Esophagus Forestomach Intestine Pancreas Liver Kidney Testis Thymus Intestinal lymph node Adult
Overall expression Neural tissues Skin Esophagus Intestine Pancreas Kidney Adrenals Testis Lymph node Lungs
mRNA
Cell type
⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹
Most cell types Purkinje cell layer Cerebral, cerebellar neurons and meningeal cells Glial cells Spinal cord and spinal dorsal root ganglion cells Ganglion, and rods and cones layers Other retinal cells Mature epithelial cells Hair follicle and sweat gland cells Conversional zone cells All cell types Squamous epithelial cells Epithelial cells Mucosal epithelial cells Serosal cells Acinar glands and islet epithelial cells All cell types Collecting tubular epithelial cells Other nephron epithelial cells Epithelial cells lining developing seminiferous tubules Thymocytes Capsular lining cells Other cell types Most tissues Cerebral hippocampus and thalamic nuclei neurons Cerebellar Purkinje cells Mature epithelial epidermal layer Hair follicle and sweat glands cells Squamous epithelial cells Mucosal and serosal cells Acinar glands and islets Collecting tubular epithelial, juxtamedullary, and glomerular cells Most cells Sertoli cells Vas deferens epithelium Capsular lining cells All cell types
Note. The mRNA signal intensity was scored as follows: ⫹, very low; ⫹⫹, low; ⫹⫹⫹, moderate; ⫹⫹⫹⫹, high.
ingly intense in all the neuronal cells compared to the embryonic brain stages where the signal was diffuse but weak. All the neuronal layers showed strong expression in the 2-day and 4-week-old postnatal mice. The more densely populated neuronal layers such as the hippocampus showed bright bands of signal in contrast to adjacent layers mostly populated by glial cell populations (Figs. 1I–L). Neuronal cells in the spinal cord and dorsal root ganglia showed similar strong expression in the postnatal mice. In addition to the neuronal cells in the CNS, strong expression was also detected in the meningeal cells (Figs. 1I and 1K) and epithelial cells of the
choroid plexus (data not shown). In the postnatal eye the retina showed strong signal in the ganglion cell layer as well as in the inner and outer nuclear layers. Differential expression was also observed in postnatal non-CNS tissues. In the skin GCase mRNA expression continued to be observed in the outer epidermal cell layers at Neonatal Day 2 and at 4 weeks (Figs. 1A and 1M, respectively). Strong signal also was observed in hair follicles (Fig. 1M). In the lymph nodes, the sinusoidal capsule showed signals of moderate intensity (Fig. 1O) with no significant signal in the lymphoid cell populations. The spleen
FIG. 1. In situ hybridization profiles of postnatal tissues using gba mRNA antisense probes. Various tissues from Neonatal Days 2– 4 (A–K) and 4-week old adult mice (L–P). (A–D) Postnatal Day 2 tissues: (A) Skin, outermost epidermal layers (arrowhead); (B) neonatal forestomach (squamous cell mucosa, arrowhead); (C) small intestine; cross section of villi (arrowhead) and serosal cell layers (arrow). (D) Bone: Signals of moderate intensity in osteoblasts (arrowhead) of bony trabeculae at the conversional zone; (E–H) Postnatal Day 4; (E) a negative control using the sense gba RNA. (F and G) Highest intensity signals throughout the mucosal epithelial cells of the small intestine (arrowheads) with moderate signals at the serosal surface. The asterisks are smooth muscle cells (negative). (H) Visceral organs:
MURINE GCase mRNA EXPRESSION
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FIG. 1—Continued
The pancreas (p), kidney tubular epithelial cells (kd), and epithelial cells lining the developing seminiferous tubules (t) and tubules of the epididymus (e) had high to moderate signals, skeletal muscle fibers (skm) show weak signal. (I–L) Various CNS regions from 2- and 4-day-old brains: (I) Cerebral cortex (layers III and V, arrowheads); (J) cerebellar cortex (Purkinje cell layer, arrowheads); (K) cerebral cortex (h, hippocampus; meninges arrowhead); (L) hippocampus (h) of a 4-week-old adult. (M–P) adult tissues: (M) skin with outer epidermal cells (arrow) and hair follicles (arrowhead); (N) squamous epithelium of the esophagus (arrowheads); smooth muscle cells (arrow) were negative; (O) lymph node (arrowhead-capsular lining cells); (P) testes (Sertoli cells; arrowhead). (All photomicrographs dark-field illumination, Panels A, C, E–G, I–L, N–P, ⫻100; Panels B, D, H, and M, ⫻40; Panel G, ⫻200).
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also showed very low expression. Expression in the bone was most intense in the conversional zones in areas of new bone growth (Fig. 1D). In contrast, little expression was observed in skeletal muscle tissue or cardiac muscle cells. Some of the most intense expression was observed in the digestive system. The squamous epithelium lining the esophagus and forestomach (Figs. 1B and 1N) showed strong signal. The epithelial cells lining the small intestine also showed strong expression (Figs. 1C, 1F, and 1G) and the colonic glandular epithelium showed moderate expression levels. The pancreas also was positive (Fig. 1H) and the liver showed only weak expression. In the genitourinary tract expression was detected in the 2-day-old newborn at weak levels in the tubular epithelium. Expression in the 4-day-old newborn kidney also was diffuse in the tubular epithelium, but slightly more intense. Expression was also observed in the more immature tubules and glomeruli in the nephrogenic zone just under the capsule (Fig. 1H). In the testis (Fig. 1P) signal was detected in the Sertoli cells lining the seminiferous tubules as well as in the epithelial cells lining the epididymus and vas deferens. DISCUSSION In situ hybridization using gba antisense RNA showed generalized low level expression early in gestation with gradual appearance of differential expression appearing around gestational age E14 and significantly increasing at term and into adulthood. During early embryonic development, except for trophoblastic cells, gba mRNA was at relatively low levels and high levels were not present in cells or regions that had high cellular turnover and/or remodeling. With increased apoptosis or remodeling during development a regional increase in gba mRNA might have been expected because of the accompanying increased metabolic demands due to membrane turnover. In the postnatal brain and mature brain increasing expression was observed in all neuronal populations throughout the brain. In contrast to the bands of strong signal observed in the neuronal layers such as the hippocampus, cortical layers, and cerebellar layers, the adjacent myelinated or myelinating regions of the CNS did not show significant levels of gba mRNA expression as had been observed with aspartylglucosaminidase (13). All variants of Gaucher disease have CNS pathology that includes periadventitial accumulations of Gaucher cells in the Virchow-Robinow spaces, and
around venules of the cerebral and cerebellar cortical regions (14,15). These cells are extraneural and probably derive from the monocyte/macrophage system secondary to the storage of GC derived from visceral sources as determined by fatty acid composition analyses (16). These cells also accumulate in a rostral to caudal gradient (17) and probably account for the majority of the similar concentration gradient of GC accumulation in brains of Type 2 and 3 patients. To understand the potential relationship between GCase expression and the pathology of Gaucher disease two different mechanisms must be considered. In the visceral tissues, the primary pathophysiology relates to the macrophage and its processing of engulfed cellular debris and old and dying cells. Such debris is derived primarily from the blood-formed elements that are phagocytosed and is present by macrophages within the liver, spleen, and bone marrow. Accumulations of GC must result from phagocytosis and the inability of the residual enzyme activity in these macrophages to completely eliminate the presented GC from glucosphingolipid precursors. This is supported by the observation of Gaucher-like cells in non-Gaucher individuals who have chronic myelogenesis leukemia. The large amounts of GC presented to macrophages is from the massive proliferation of myelocytic cells whose glucosphingolipids require processing through the sequential hydrolytic system (18,19). The mass of this substrate presentation overwhelms the degradative capacity of even normal amounts of enzyme and Gaucher-like cells accumulate. It might be anticipated that if larger amounts of functional GCase were present in these normal cells that such cells would not accumulate GC. Thus, some imbalance of substrate presentation (glucosylceramide flux) and GCase amount in cells is essential for substrate accumulation and Gaucher cell development in visceral tissues. Interestingly, higher levels of gba mRNA expression were found in a variety of epithelial cells in visceral organs, including the skin. These epithelial cells are desquamated and are eliminated from the body. Thus, they do not have the chance to accumulate substrate, even if it could be phagocytosed and exceeded the capacity of the residual activity to catabolize this material. Similarly, the nonphagocytic cells of the liver and lymphoid system do not accumulate significant amounts of GC in Gaucher disease since the normal amount of presented GC likely derives from endogenous cellular synthesis and does not exceed the degradative capacity of those cells.
MURINE GCase mRNA EXPRESSION
The pathophysiology in the brain is different since neuronal involvement and neuronal death are major events that lead to degeneration. Excluding the accumulations of macrophages (peripherally derived) in the Virchow-Robinow spaces, a gradient of involvement of neurons from rostral to caudal is not apparent in the brains of affected Type 2 patients (5,17). However, regional variation of neuronal cell loss does occur. This includes more distinct involvement of layers III and V in the cerebral cortex, hippocampal pyramidal cells, neurons of the thalamic and basal ganglia, and the dentate nucleus of the cerebellum. These areas show the highest degrees of neuronal cell loss with subsequent gliosis. Based on the mRNA expression, gba appears uniformly throughout the CNS. Regional variation may relate to species differences between humans, where the pathology has been described and the expression pattern described in the mouse brain. Alternatively the regional differences described in the pathology of human gba deficiency may be the result of multiple factors such as differences in metabolic activity, vascular perfusion, or other contributing factors which may make certain neuronal populations more susceptible to degeneration as a result of gba deficiency. Previously, glucosphingosine, the deacylated analogue of GC, was suggested, in analogy to galactosphingosine in Krabbe disease, as the toxic compound leading to neuronal death (20). Although this remains a viable pathophysiologic explanation, direct proof is lacking. The toxicity of accumulated GC has not been excluded and, indeed, the inflammatory reaction surrounding some of the accumulated macrophages in the Virchow-Robinow spaces (21) and the overexpression of macrophage genes in Gaucher cells (22) suggest the possibility of direct involvement and/or toxicity of GC in the pathophysiologic process of the central nervous system and visceral involvement in neuronopathic Gaucher disease.
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ACKNOWLEDGMENT This work was supported by NIH Grant RO1 DK 36729 (G.A.G.).
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