Mice Deficient in Lysosomal Acid Phosphatase Develop Lysosomal Storage in the Kidney and Central Nervous System*

(Received for publication, March 31, 1997, and in revised form, May 20, 1997)

Paul Saftig Dagger §, Dieter Hartmann , Renate Lüllmann-Rauch , Joachim Wolff par , Meike Evers Dagger **, Anja Köster Dagger Dagger Dagger , Michal Hetman Dagger §§, Kurt von Figura Dagger and Christoph Peters Dagger **

From the Dagger  Zentrum Biochemie und Molekulare Zellbiologie, Abteilung Biochemie II, Universität Göttingen, Gosslerstrasse 12D, 37073 Göttingen, the  Anatomisches Institut, Christian Albrechts Universität Kiel, 24118 Kiel, and the par  Zentrum Anatomie, Universität Göttingen, 37075 Göttingen, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Lysosomal acid phosphatase (LAP) is a tartrate-sensitive enzyme with ubiquitous expression. Neither the physiological substrates nor the functional significance is known. Mice with a deficiency of LAP generated by targeted disruption of the LAP gene are fertile and develop normally. Microscopic examination of various peripheral organs revealed progredient lysosomal storage in podocytes and tubular epithelial cells of the kidney, with regionally different ultrastructural appearance of the stored material. Within the central nervous system, lysosomal storage was detected to a regionally different extent in microglia, ependymal cells, and astroglia concomitant with the development of a progressive astrogliosis and microglial activation. Whereas behavioral and neuromotor analyses were unable to distinguish between control and deficient mice, ~7% of the deficient animals developed generalized seizures. From the age of 6 months onward, conspicuous alterations of bone structure became apparent, resulting in a kyphoscoliotic malformation of the lower thoracic vertebral column. We conclude from these findings that LAP has a unique function in only a subset of cells, where its deficiency causes the storage of a heterogeneously appearing material in lysosomes. The causal relationship of the enzyme defect to the clinical manifestations remains to be determined.


INTRODUCTION

Lysosomal acid phosphatase (LAP1; EC 3.1.3.2) is an orthophosphoric monoesterase of the endosomal/lysosomal compartment (1, 2). Like the highly homologous secretory acid phosphatase of the prostate (3), LAP is sensitive to inhibition by L-tartrate (4). In vitro, LAP cleaves various phosphomonoesters (e.g. adenosine monophosphate, thymolphthalein phosphate, and glucose 6-phosphate) at an acidic pH (3.5-5.0) (4, 5). Despite the pivotal role LAP has played in the discovery and histochemical visualization of lysosomes (6), neither its in vivo substrates nor its functional role is known.

The biosynthetic route of LAP is quite unusual since it is synthesized and transported as an integral type I membrane glycoprotein with a large luminal domain containing the active site of the enzyme, a single transmembrane domain, and a short cytoplasmic domain (7). The LAP precursor is initially transported to the plasma membrane, from where it is rapidly internalized by receptor-mediated endocytosis via clathrin-coated pits (8, 9). Before its eventual delivery to the lysosomes, the membrane-bound LAP precursor repeatedly recycles from the early endosomes to the cell membrane (8). The cytoplasmic domain of the LAP precursor harbors a tyrosine-containing internalization signal (10, 11) as well as a signal for basolateral sorting (12). The membrane-bound LAP precursor, which forms homodimers in vivo, is converted to soluble mature LAP after delivery to the lysosomal compartment by limited proteolysis (13).

In situ hybridization showed an almost uniform expression of LAP in all organs of the mouse, except for the testis and brain, which stand out through their considerably higher level of expression. In the testis, the higher expression is restricted to spermatocytes, whereas in the brain, expression in cerebellar Purkinje cells and telencephalic pyramidal neurons as well as in epithelial cells of the choroid plexus predominates (14).

Besides tartrate-sensitive LAP, the tartrate-resistant type 5 acid phosphatase (TRAP), another acidic orthophosphoric monoesterase, has been identified in the lysosomal compartment (15). TRAP enzyme activity has been detected in the mononuclear phagocyte system, i.e. alveolar macrophages and osteoclasts (16). The functional relationship between both phosphatases is still unknown.

To elucidate the physiological role of LAP, we have inactivated the corresponding gene in the mouse by targeted disruption. In this study, we describe the phenotype of LAP-deficient mice, which is characterized by lysosomal storage phenomena in the kidney and central nervous system, progressive skeletal disorder, and an increased disposition toward generalized seizures.


EXPERIMENTAL PROCEDURES

Isolation of a Genomic Clone and Construction of a Target Vector

A C57BL/6J mouse cosmid library (Stratagene, La Jolla, CA) was screened with a full-length mouse LAP cDNA clone (17). The isolated mouse LAP cosmid clone mLAP1 contained all 11 exons of the gene as well as several kilobase pairs of the 5'- and 3'-flanking regions. DNA sequence analyses revealed complete sequence identity of exon sequences to the nucleotide sequence of the mouse LAP cDNA used for screening (data not shown). For construction of a target vector, a 9-kbp HindIII DNA restriction fragment of mLAP1 covering 3.7 kbp of 5'-untranslated region and exons 1-8 (see Fig. 1A, panel I) was subcloned into the plasmid vector pBluescript SK II+ (Stratagene). The neo expression cassette from pMC1neopA (Stratagene) (18) was inserted as a BglII DNA restriction fragment into a BamHI restriction site located in exon 7 of the LAP gene. The insertion of the neo cassette introduces a premature translational stop codon into the open reading frame of the LAP gene.


Fig. 1. Disruption of the LAP gene. A, strategy for disruption of the LAP gene by homologous recombination in mouse ES cells. Panel I, genomic structure and partial restriction map of the LAP gene. Exons are numbered and indicated by open boxes. Exon 7, used for interruption of the open reading frame, is indicated by the closed box. Introns and 5'- and 3'-flanking regions are indicated by the solid line. The black bar designated probe A denotes the DNA probe used for Southern blot analyses. Panel II, the target vector pLAP-9HIIIneo with 9.0-kilobase homology to the LAP gene locus. The neo cassette (open box) was inserted into a BamHI restriction site located in exon 7; the arrowhead marks the direction of transcription of the neo gene. The broken line indicates the plasmid vector pBluescript SK II+. Panel III, predicted LAP gene locus after homologous recombination. B, BamHI; H, HindIII; S, SstI; X, XbaI. B, Southern blot analyses of ES cell clones. Panel I, probe A was hybridized to SstI-digested DNA from wild-type E-14-1 ES cells and two ES cell clones (EL-5 and EL-178) with a targeted allele as indicated by an additional 4.6-kbp DNA fragment. Panel II, probe A was hybridized to XbaI-digested DNA from the same ES cells (as in panel I), confirming homologous recombination in EL-5 and EL-178 as indicated by an additional 4.2-kbp fragment. C, Southern blot analysis of tail DNA. DNA was digested with the restriction enzyme SstI and hybridized with probe A (wild-type allele: 3.4 kbp; mutant allele: 4.6 kbp). +/+, homozygous wild-type; +/-, heterozygous; -/-, homozygous mutant.
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Selection of Targeted Embryonic Stem (ES) Cells and Generation of Mutant Mice

The recombination construct (pLAP-9HIIIneo; see Fig. 1A, panel II) was linearized with XhoI and introduced into the ES cell line E-14-1 by electroporation. ES cells were cultured as described previously (19, 20). G418-resistant colonies were screened by Southern analysis of genomic DNA digested with SstI or XbaI and probed with probe A (see Fig. 1A, panels I and III). Mutated ES cells were microinjected into blastocysts of C57BL/6J mice as described (20). The resulting male chimeras were mated with C57BL/6J females. Mice were genotyped for the introduced LAP gene mutation by Southern analysis of SstI-digested genomic DNA using probe A (see Fig. 1A, panels I and III). The mice were kept in a conventional animal facility at the Zentrum für Biochemie und Molekulare Zellbiologie, Universität Göttingen (Göttingen, Germany).

Northern Blot Analysis

Total RNA from the livers and kidneys of 3-month-old mice was prepared as described by Chirgwin et al. (21). Ten micrograms of total RNA were separated on a formaldehyde-agarose gel and processed as described (22). Filters were hybridized with the mouse LAP cDNA (17) and a 280-base pair cDNA fragment from glyceraldehyde-3-phosphate dehydrogenase (23). Hybridization and washing of filters were performed as described (10).

LAP Assay

Spleen and liver tissues were homogenized in 10 mM Tris-HCl and 150 mM NaCl, pH 7.4 (w/v, 1:3), at 4 °C. Homogenates were adjusted to 0.1% Triton X-100, sonicated for 30 s on ice, and centrifuged at 12,000 × g for 5 min at 4 °C. The protein concentration of the supernatant was determined (24). Aliquots of the supernatant were incubated with 100 µl of 10 mM p-nitrophenyl phosphate in 0.1 M sodium citrate, pH 4.5, and either 50 µl of 40 mM NaCl or 50 µl of 40 µM tartrate. After incubation for 20 min at 37 °C, the reaction was stopped by the addition of 0.5 ml of 0.4 M glycine/NaOH, pH 10.4. Absorbance at 405 nm was determined. The tartrate-inhibitable LAP activity was determined as described (1).

Tissue Preparation for Morphological Analysis

For standard light microscopic histology and immunohistochemistry, brains were fixed by cardiac perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer and shock-frozen or fixed by perfusion with Bouin's solution diluted 1:3 with 0.1 M phosphate buffer and embedded in paraffin. Series of frozen sections were processed for lectin histochemistry using Griffonia simplicifolia agglutinin (GSA) coupled to peroxidase (Sigma, Deisenhofen, Germany) at a concentration of 5 mg/ml, followed by enzyme histochemistry of peroxidase according to Adams (25). Alternating sections were immunohistochemically reacted with monoclonal antibodies against lamp-1 (lysosome-associated membrane protein-1; 3 mg/ml; Developmental Studies Hybridoma Bank, University of Iowa) or cathepsin D-specific antiserum (26) in a 1:1000 dilution in phosphate buffer following the peroxidase/anti-peroxidase method of Sternberger (27). Similarly, antibodies (mono- and polyclonal) against tyrosine hydroxylase (Chemicon International, Inc.), glutamate decarboxylase (a gift from W. Oertl), phosphorylated and dephosphorylated neurofilaments (Sternberger-Meyer Immunocytochemistry Inc.), GAP-43 (Sigma), GFAP (glial fibrillary acidic protein) (Sigma), S100 (DAKO, Hamburg, Germany), tenascin (a gift from M. Schachner), c-Fos (Oncogene Science Inc.), and Ulex europaeus agglutinin I lectin (Sigma) were applied using standard histochemical techniques.

To visualize the distribution of some antigens with better tissue preservation, paraffin sections were dewaxed and reacted with antibodies against GFAP, lamp-1, cathepsin D, and MHC II (Pharmingen, Hamburg) and the microglia-specific lectins GSA-I-B4 and Ricinus communis agglutinin I (RCA-I), the latter one predominantly reacting with activated microglia. Binding of both lectins and primary antibodies was detected by tyramide amplification (reagents purchased from NEN Life Science Products) developed by peroxidase histochemistry (28, 29).

For electron microscopy, the fixative was modified by adding 0.5% glutardialdehyde, while the histochemical procedures remained unchanged. Finally, tissue sections were treated with 2% osmium tetroxide, dehydrated, and embedded in Epon 812. For standard transmission electron microscopy without the application of histochemical procedures, animals were perfused with 6% glutardialdehyde, and tissues were embedded in Araldite.

Radiographs

Mice were briefly narcotized to facilitate radiography. For comparative purposes, high resolution Eastman Kodak mammography x-ray film was employed, and identical radiation energy (35 kV) and photographic exposures were used to prepare radiographs on separate portions of the same film.


RESULTS

Generation of Mice with Targeted Disruption of the Lysosomal Acid Phosphatase Gene

A mouse genomic DNA clone covering the entire LAP gene was isolated from a C57BL/6J mouse cosmid library (for details, see "Experimental Procedures"). The target construct pLAP-9HIIIneo (Fig. 1A, panel II) was used for disruption of the LAP gene in ES cells. The open reading frame of the gene is interrupted in exon 7, and the truncated open reading frame encodes 243 of 423 amino acids of wild-type LAP. Since conversion of histidine 256 of human LAP, which is conserved among lysosomal, prostatic, yeast, and Escherichia coli acid phosphatases and was proposed to be part of the active sites of these enzymes (30), to serine yielded a catalytically inactive LAP,2 the introduced mutation is most likely a null mutation.

pLAP-9HIIIneo was introduced into E-14-1 ES cells (31), and G418-resistant colonies were analyzed by Southern blotting. In 3 out of 532 independent clones tested, an additional SstI DNA restriction fragment was detected with probe A (Fig. 1A), indicating a homologous recombination event in one of the LAP alleles (Fig. 1B, panel I). This was confirmed by probing XbaI-digested DNA with probe A (Fig. 1B, panel II). The targeted ES cell clones were microinjected into C57BL/6J blastocysts. The resulting chimeras were used to generate heterozygous and subsequently homozygous mutant offspring in an outbred 129SVJ-C57BL/6J genetic background.

Heterozygous offspring identified by hybridization of SstI-digested DNA with probe A (Fig. 1C) did not show differences in phenotype or fertility as compared with wild-type littermates (data not shown). Genotyping of 92 offspring from heterozygous crosses revealed a frequency of 24% for homozygous mutant mice, closely resembling the expected mendelian frequency (25%). Hence, disruption of the LAP gene does not result in embryonic lethality.

Functional Inactivation of the Lysosomal Acid Phosphatase Gene

To test for expression of LAP at RNA and enzyme activity levels, Northern blot analyses and acid phosphatase enzyme assays were performed. A 3.2-kilobase LAP-specific RNA hybridized in liver RNA from a wild-type animal, whereas no LAP transcripts were detectable in liver RNA from a homozygous mutant animal (Fig. 2A). For determination of LAP enzyme activity, spleen and kidney homogenates were incubated with p-nitrophenyl phosphate in the presence and absence of L-tartrate, a competitive inhibitor of LAP. LAP-specific, L-tartrate-sensitive enzyme activity was undetectable in homogenates from homozygous mutant animals (Fig. 2B). Northern blot analyses as well as LAP enzyme activity assays showed that the LAP gene had been inactivated and that homozygous mutant mice were devoid of lysosomal acid phosphatase activity.


Fig. 2. The LAP gene is inactivated in homozygous mutant mice. A, Northern blot analysis of LAP expression. Total RNA (10 µg) was hybridized using a LAP mouse cDNA probe (17) and a murine glyceraldehyde-3-phosphate dehydrogenase (G3PD) probe (internal control) (23). LAP mRNA (3.2 kilobases) was absent in the livers and kidneys of homozygous mutant mice (results shown for the liver only). B, LAP enzyme activity. Spleen and kidney homogenates from homozygous mutant mice (-/-) and wild-type controls (+/+) were incubated with p-nitrophenyl phosphate in the presence and absence of 50 mM L-tartrate. The tartrate-inhibitable enzyme activity was determined. No LAP enzyme activity was found in LAP-deficient tissues (n = 2; range is given).
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Lack of Lysosomal Storage Material in Lysosomal Acid Phosphatase-deficient Fibroblasts

To identify phosphorylated storage material in LAP-deficient cells, embryonic fibroblasts were metabolically labeled with [32P]orthophosphate. After subcellular fractionation by Percoll density centrifugation, the fractions enriched in markers for lysosomes or endosomes were subjected to analysis for low molecular weight, water-soluble material by gel- and ion-exchange chromatography; for polypeptides by two-dimensional electrophoresis; and for lipids by extraction with chloroform/methanol and thin-layer chromatography. None of these experimental procedures revealed the accumulation of 32P-labeled material in LAP-deficient fibroblasts (data not shown).

Phenotype of Lysosomal Acid Phosphatase-deficient Mice

Homozygous mutant, heterozygous, and wild-type mice resulting from heterozygous crosses did not exhibit differences in growth and weight development (data not shown). LAP-deficient mice were fertile and did not show an elevated mortality up to an age of 18 months. Gross anatomy and both light and electron microscopic observations of tissues such as liver, spleen, muscle, retina, and thyroid gland of 3-9-month-old mice showed no significant differences compared with control mice. After the age of 6 months, alterations of bone structure became apparent, resulting in a kyphoscoliotic malformation of the lower thoracic and lumbar vertebral column (see Fig. 7).


Fig. 7. Radiologic examination of LAP+/+ and LAP-/- mice at 6 (A and B) and 15 (C-F) months of age. A and B, after an apparently normal development of the skeletal system, pathological alterations were first noted at the age of 6 months, when LAP-deficient mice (B) exhibited an augmented lower thoracic kyphosis and an upper thoracic lordosis of the vertebral column (arrowheads in B) as compared with controls (A). At this stage, the overall bone structure appears otherwise normal. C-F, until 15 months of age, LAP-deficient mice (D and F) have developed a considerable S-shaped deformation of the entire thoracic vertebral column and severe alterations of bone structure. Specifically, individual vertebral bodies feature an increased sagittal diameter (short arrows in E and F), whereby individual vertebral bodies exhibit a reduced axial height (indicated by arrowheads for LIII). In LAP-deficient mice, the pediculi of vertebral arches extend over almost the entire length of the corpus vertebrae (long arrows), thus leading to a dramatic reduction in size of the intervertebral foramina (small arrows pointing to intervertebral foramen LIV) and possibly to a compression of the corresponding spinal nerves. Control mice are shown in C and E.
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Lysosomal Storage in the Kidney

Among the peripheral organs analyzed, lysosomal storage was observed only in the kidney. Ultrastructural examination revealed enlarged lysosomes in some podocytes (Fig. 3A) and those portions of Henle's loop that are located within the inner stripe of the outer medulla, i.e. the uppermost portion of the thin descending limbs (Fig. 3, B and D) and the thick ascending limbs. In wild-type mice, the podocytes (data not shown) and the cells of Henle's loop (Fig. 3C) show only very few small lysosomes. In LAP-deficient mice, the lysosomes were increased in size and number and contained multilamellar or polymorphic material.


Fig. 3. Lysosomal storage in LAP-deficient kidney. A, podocyte with a storage vacuole (arrow) containing flocculent material. B, epithelial cell of an intermediate tubule. The lysosomes contain multilamellar and granular materials (arrows). C and D, light micrographs of intermediate tubules of a control (C) and a LAP-deficient (D) mouse. The storage lysosomes appear as intensely staining cytoplasmic inclusions (arrows). Bars = 0.5 µm (A), 0.25 µm (B), and 10 µm (C and D).
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Neuropathology of LAP-deficient Mice

When analyzed with a variety of standard techniques such as Nissl and hematoxylin/eosin staining and incubation with antibodies to neuronal growth cones, synaptophysin, phosphorylated and nonphosphorylated neurofilaments, dopaminergic and GABAergic neurons and olfactory axons, S100 protein (glial and neuronal marker), and tenascin (predominately a glial product), no differences between LAP-deficient and control brains were observed (data not shown) in brain sections of mice 3-11 months of age. To provide a first survey of lysosomal alterations, these organelles were detected by immunohistochemical labeling of the corresponding marker antigens lamp-1 and cathepsin D. The immunoreactivity for lamp-1 was evenly distributed at a comparatively low level in control mice (Fig. 4, A, C, and E), whereas antibodies to cathepsin D strongly labeled the somata of all kinds of neurons (shown for control cerebral cortex in Fig. 4G).


Fig. 4. Enhanced immunoreactivity of LAP-deficient glial cells for lamp-1 and cathepsin D. Shown is the immunohistochemical distribution of the lysosomal marker lamp-1 in parasagittal sections of control (A, C, and E) and LAP-deficient (B, D, and F) mouse brain. A and B, cerebral cortex. In superficial cortical layers of normal brains (A), the immunoreactivity for lamp-1 is weak, whereas in the LAP-deficient cerebral cortex (B), the glial cells of the lower half of the cortex show a strong labeling for lamp-1. C and D, striatum. At higher magnification, only some glial cells of the normal striatum (C) are faintly stained, whereas in the LAP-deficient striatum (D), the number of strongly stained glial cells, both microglia and astroglia, is higher than in controls. E and F, cerebellar cortex. In the cerebellar cortex of control brains (E), immunoreactivity is concentrated in the Purkinje cell layer (pl). Here the neurons (marked by arrows) are less stained than cell bodies of Bergmann glia (bg). In LAP-deficient mice (F), Bergmann glial cells (arrows), subpopulations of astrocytes (a) in the granular layer (gl), and microglial cells (m) in the granular and molecular layers are strongly labeled. G and H, cerebral cortex. The immunoreactivity for cathepsin D in laminae V and VI of the dorsal isocortex is shown for controls (G) and LAP-deficient mice (H). Note the intense staining of neuronal perikarya in controls, which is almost lost in LAP-deficient animals. Bars = 100 µm (A and B), 20 µm (C and D), 50 µm (E and F), and 200 µm (G and H).
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In LAP-deficient mice, strongly lamp-1-immunoreactive glial cells appeared in deep layers of the cerebral cortex (Fig. 4B). At higher magnification (shown for the striatum in Fig. 4, C and D), it was apparent that the weak staining of glial cells in control mice was replaced by a strong staining of the cell bodies of glial cells in LAP-deficient mice. In the cerebella of LAP-deficient mice, a prominent staining for lamp-1 was seen in Bergmann glial cells, subpopulations of astrocytes in the granular layer, and microglial cells in the granular and molecular layers (Fig. 4F), whereas in control mice, immunoreactivity for lamp-1 was weak and mainly confined to Bergmann glia (Fig. 4E). In addition, in LAP-deficient mice, antibodies to lamp-1 strongly labeled ependymal cells of the ventricular lining with a gradient of decreasing intensity from the spinal chord central canal to the hemispheral ventricles (data not shown).

In LAP-deficient mice, immunoreactivity for cathepsin D as a second lysosomal marker protein was enhanced in most brain regions due to staining of enlarged microglial and astroglial cell bodies and globular structures within their processes (data not shown). Interestingly, cathepsin D immunoreactivity tended to be reduced in neurons (Fig. 4H).

Routine immunohistochemistry for the glial marker GFAP revealed a localized astrogliosis restricted to those brain regions that showed increased lamp-1 staining. Thus, strongly GFAP-positive astroglia cells were found in the deep layers (V/VI) of the isocortex, basal ganglia, and cerebellar Purkinje cell layer (data not shown).

Electron microscopic analysis of LAP-deficient mice showed that the majority of astrocytes, cerebellar Bergmann glial cells, and ependymal cells contained numerous storage vacuoles, which could be identified as lysosomes by staining with lamp-1 (shown for astroglia in Fig. 5B) and cathepsin D (data not shown) antibodies. Conventional electron microscopy revealed that the storage vacuoles either appeared electron-lucent or were filled with material of variable electron density (shown for Bergmann glia in Fig. 5D and ependymal cells in Fig. 5F). In control mice, lysosome-like structures were barely detectable in astrocytes, Bergmann glia, and ependymal cells (Fig. 5, A, C, and E). Lysosomal storage in glial cells of LAP-deficient brains appeared to be a progredient process and also exhibited a gradient of decreasing severity from distal to proximal parts of the brain.


Fig. 5. Lysosomal storage in LAP-deficient macroglial cells. Shown are electron micrographs of macroglial cells from control (A, C, and E) and LAP-deficient (B, D, and F) mice. A and B, astroglial cells. Immunoelectron microscopy for lamp-1 reveals labeling of a lysosome-like structure (arrow) in the control astrocyte (A), but a strong labeling of storage vacuoles (arrows) in the LAP-deficient astrocyte (B). C and D, Bergmann glial cells. Conventional electron microscopy of Bergmann glial cells demonstrates storage vacuoles (arrowheads) in LAP-deficient animals (D), which were absent in controls (C). The storage vacuoles are partly filled with heterogeneously appearing material. E and F, ependymal cells. Conventional electron microscopy of ependymal cells reveals lysosomal storage (arrow) in LAP-deficient ependymal cells (F), which was not found in controls (E). Bars = 1 µm (A and B), 0.5 µm (C and D), and 1.5 µm (E and F).
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Similar to astroglia, microglial cells exhibited conspicuous alterations within LAP-deficient brains. Thus, in control mice, staining of microglial cells with B4 isolectin from G. simplicifolia seeds (GSA) gave a rather uniform staining pattern throughout the brain (Fig. 6A). This alpha -D-galactosyl-specific lectin has been shown to selectively label endothelia and microglial cells in rodents (32). In control mice, the lectin outlined the extensive processes of microglial cells (Fig. 6C). Electron microscopy revealed that binding of GSA was confined to the cell surface and a few lysosome-like structures (Fig. 6E, arrows). A remarkably enhanced staining was found when brain sections of LAP-deficient mice were stained with GSA. In LAP-deficient mice, the staining intensity of microglial cells was increased in almost the entire brain. The GSA staining pattern closely mimicked that obtained with lamp-1. As for astroglia, the deep isocortical laminae, basal ganglia, cerebellum, and brain stem were most intensely labeled, whereas other areas such as the superficial layers of the neocortex (laminae I-II), the first layer of the paleocortex, and the dorsal segment of the hippocampus (Fig. 6B, stars) remained unchanged with respect to control mice. The increased staining intensity of microglial cells was due to an enhanced labeling of their cell bodies (Fig. 6D), which were filled by GSA-positive vesicles (Fig. 6F, arrows). Conventional electron microscopy showed that the vacuoles were filled with material of variable electron density, which apparently was lost during the sample preparation for lectin staining (data not shown). As revealed by immunohistochemistry for lamp-1 and cathepsin D, the vacuolar storage granules of microglial cells from LAP-deficient mice (shown for cathepsin D in Fig. 6, G and H, arrows) were of lysosomal origin.


Fig. 6. Microglial cells in LAP-deficient mice contain cytoplasmic vacuoles that stain with GSA and cathepsin D. A and B, parasagittal sections of 3-month-old mouse brains. At low magnification, a uniform and weak staining is observed with GSA throughout the control brain (A), with somewhat stronger staining along marginal and paraventricular brain surfaces. In a 3-month-old LAP-deficient mouse brain (B), enhanced GSA staining is based on a spotty staining pattern in most brain parts. Zones with weak staining are labeled (*) in superficial cortex layers. The strongest staining is observed in the anterior parts of the striatum (s) and cerebral cortex (c) as well as in some parts of the hippocampus (h). C and D, deep cortical layers. At higher magnification, GSA labels normal microglial cells at variable density in deep cortical layers (C). At higher magnification in LAP-deficient brains (D) (picture shows a comparable part of the deep cortical layer in C), the cell bodies of almost all microglia cells are strongly labeled with GSA, whereas labeling appears equal or even reduced in many microglial cell processes. E and F, microglial cells. Electron microscopy shows that in control animals (E), GSA labels selectively the surface of microglial cells and a few vacuolar, lysosome-like structures (arrows). Electron microscopy of microglial cells of LAP-deficient mice (F) shows a prominent GSA labeling of polymorphic vacuole membranes, whereas labeling of the cell surface is sparse or absent. Labeled vesicular structures are indicated by arrows. G and H, microglial cells. Immunoelectron microscopy for cathepsin D shows that in controls (G), weak labeling of the microglial cell surface (arrows) is observed, whereas in LAP-deficient cells (H), labeling of the limiting membrane of cytoplasmic vacuoles is prominent and that of the cell-surface is scarce. Bars= 1 mm (A and B), 10 µm (C and D), 1 µm (E and F), and 0.5 µm (G and H).
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Up-regulation of GSA-binding sites is regarded as one of many parameters characterizing microglial cell activation. Binding sites for the lectin RCA-I, which are present only at low levels in resting microglia, were strongly expressed by microglia of LAP-deficient mice in the same brain regions that were distinguished by the most intense up-regulation of GSA-binding sites. Thus, cells labeled by RCA-I were scattered e.g. along the isocortical lamina V and the cerebellar Purkinje cell layer (data not shown).

In younger LAP-deficient animals, both lectins stained cells of the already activated, but still ramified phenotype. A change in microglial morphology toward an epithelioid (phagocytic) morphology was observed at later stages (>8 months), which was then encompassed by the expression of MHC II antigens by a subset of these cells. However, other markers of microglial activation (NADH diaphorase and nitrogen-monoxide synthetase) were not elevated in deficient animals (data not shown).

Neurological Examination

Among 270 LAP-deficient mice, 19 animals (7%) repeatedly presented epileptic seizures at >8 weeks of age. These seizures were generalized and tonic-clonic in nature. They were triggered by the weekly cage changing and followed by a short period of excitement caused by placement of the animal in a new environment. The convulsions lasted between 5 and 30 s, during which the animal lost consciousness and balance. The seizures were followed by 5-15 min of postepileptic depression, in which the animal did not respond to external stimuli. Increased susceptibility to auditory or electric shock provocation was not observed.3 In about the same number of wild-type mice with the same genetic background, seizures were not observed. Behavioral tests including the open-field test (33), rotarod test, wire test, and hot-plate test (34) performed at an age of 3 months failed to distinguish between LAP-deficient and wild-type control mice (data not shown).

Skeletal Malformations in LAP-deficient Mice

LAP-deficient animals of later adult stages developed conspicuous and progressive alterations of bone structure already visible in x-ray imaging whereby lesions were most prominent in vertebrae and long bones of the upper and lower limbs (shown for 6- and 15-month-old animals in Fig. 7). Specifically, areas of compact bone as well as the endplates of vertebral bodies appeared less electron-dense, whereas regions of cancellous bone (again most notably within vertebral bodies) featured an abnormally coarse and locally hyperdense meshwork of mostly longitudinally oriented trabeculations. As a consequence, the clear-cut radiographic differences between cancellous and compact bone were almost lost.

Concomitant to this apparent reduction in calcium salts within compact bone, a progressive change in bone morphology was noted, which was most prominent in thoracic and lumbar vertebrae. Thus, at the age of 15 months (Fig. 7, C-F), the individual vertebral bodies were thickened along their dorsoventral axis and also exhibited a reduced craniocaudal height (Fig. 7, E and F, short arrows and arrowheads). Vertebral arches had stunted pediculi that extended over almost the entire length of the vertebral bodies (Fig. 7, E and F, long arrows), thereby leading to a dramatic reduction in the size of the intervertebral foramina, possibly causing a generalized compression of spinal nerves (Fig. 7F). Within the thoracic vertebral column, animals of this age exhibited a scoliotic malformation with an augmented lower cervical lordosis and lower thoracic kyphosis. Interestingly, the alterations described developed comparatively late. Thus, at the age of 6 months (Fig. 7, A and B), only a slight augmentation of the lordotic and kyphotic bends of the lower cervical and thoracic vertebral column was noted.


DISCUSSION

Deficiency of LAP Causes Tissue-specific Lysosomal Storage

Even though L-tartrate-sensitive LAP is the enzyme that led De Duve (35) to the discovery of lysosomes, its in vivo functions remained elusive until today. The gene coding for LAP has been shown to be a housekeeping gene, and LAP activity has been found in all tissues and nucleated cells that have been examined (1, 14). If LAP is critical for the catabolism of one or several of its phosphorylated substrates, we reasoned that a deficiency of LAP would cause the accumulation of these substrates in lysosomes. In analogy to the >30 different lysosomal storage disorders caused by the inactivity of one or several lysosomal proteins (36), we expected that this accumulation would lead to the morphological, biochemical, and clinical manifestation of a lysosomal storage disorder.

In the early seventies, patients in two unrelated families were described that were characterized by a complete deficiency of phosphatase activity in lysosomes. The familial occurrence of the disease and the intermediate phosphatase activities in the parents suggested an autosomal recessive inheritance of the disease and an essential role of an acidic phosphatase in lysosomal catabolism (37, 38). The relation of the acid phosphatase deficiency to the clinical manifestations (progressive lethargy, opisthotonus, bleeding, and death within the first year of life) and the molecular basis remained speculative, and no further patients have been described.

Among the tissues from LAP-deficient mice investigated, lysosomal storage phenomena were observed only in podocytes and distinct segments of the nephron in the kidney as well as microglia and different subtypes of macroglial cells within the brain. In all cases, the morphology of the stored material was heterogeneous, possibly indicating the presence of different substrates in different cells, which in turn would concur with the broad spectrum of substrates cleaved by LAP in vitro. All other tissues were free of morphological signs of lysosomal storage. Moreover, the search for storage material in LAP-deficient fibroblasts with biochemical methods remained elusive. This indicates that LAP is not critical for the lysosomal catabolism in these tissues either because the substrates of LAP have a tissue-specific occurrence or because the function of LAP can be passed by alternative lysosomal phosphatases or transporters that allow the exit of phosphorylated compounds from the lysosomal compartment.

Lysosomal Storage in Glial Cells

Storage vacuoles were found in astroglia, cerebellar Bergmann glia, ependymal cells, and microglial cells. They were identified at the ultrastructural level as lysosomes by their immunoreactivity for the lysosomal membrane marker lamp-1 and the lysosomal matrix marker cathepsin D. The material contained in these storage lysosomes was of heterogeneous morphological appearance and remains to be identified. It is not clear whether the stored material originates from the glial cells or is taken up from the surrounding tissue. It should be noted that the storage in astrocytes and microglial cells is not uniform, but shows clear regional differences.

In addition to lysosomal storage, the microglial cells display characteristics of a progressive activation, i.e. the enhanced expression of binding sites for GSA and RCA-I and up-regulation of the MHC II molecules during later stages of development. At present, it is not known if the lysosomal storage per se or if signals from the surrounding milieu trigger the observed microglial activation.

LAP-deficient Mice Exhibit an Increased Disposition toward Generalized Seizures

The lysosomal storage in restricted cells might functionally be related to neuronal and skeletal alterations in LAP-deficient mice. The molecular correlation between the observed functional changes and the LAP deficiency is still unknown and deserves further investigations.

Contrasting to the moderate extent of neuropathologic damage observed in LAP-deficient mice, these animals repeatedly presented generalized tonic-clonic seizures at >8 weeks of age. The low penetrance of these seizures (~7% of LAP-deficient mice) may be linked to the genetic background of the animals investigated. It has been described earlier that C57BL/6J mice, which provide half of the 129SVJ-C57BL/6J outbred background used in this study, are very resistant to epileptic seizures at all ages (39, 40). The causal relationship between the predominantly glial pathology and the development of epileptic seizures remains unexplained. The astroglia has repeatedly been involved in the spreading of epileptic potentials by its ion transport and buffering capabilities (41). Thus, one might speculate that the lysosomal storage impairs the buffering capacity of glial cells and thereby facilitates generation of seizures. Furthermore, activated microglia may contribute to ictogenesis through secretion of neurotoxic agents that increase glutamate receptor-mediated excitotoxicity (42).

Skeletal Abnormalities

For most of the clinical manifestations in lysosomal storage disorder, the link between the molecular defect and the phenotypic alteration is not understood. This applies also for the alterations of bone structure observed in LAP-deficient mice, of which the decreased mineralization observed from the age of 6 months is the most notable feature. According to data from human bone pathology, a loss of calcium salts from bones has to be quite substantial (~30%) to be detected by standard radiographic procedures as employed here (43). It is therefore likely that the decreased mineralization of compact bone and vertebral endplates would be detectable much earlier with more sensitive methods. The coarse structure of cancellous bone especially noted in vertebrae can be interpreted as a compensatory reaction to the apparent loss of minerals from the compacta and the resulting destabilization. The foreshortening of individual bones indicates that this compensation remains inadequate.

Contrasting to frequently observed perturbations of enchondral ossification in lysosomal storage diseases (44), alterations of enchondral ossification have not been detected so far in LAP-deficient animals, and skeletal abnormalities were found only at later stages. A defective enchondral ossification with delayed mineralization of the cartilage was observed in 6-8-week-old mice with a deficiency of TRAP (45). TRAP is also localized in the lysosomal compartment (15). The expression of TRAP, however, is limited to the mononuclear phagocyte system (16). In older mice with a TRAP deficiency, an osteopetrosis with increased mineralization of bone tissue was observed.

The different phenotypes of LAP- and TRAP-deficient mice clearly indicate that the two lysosomal phosphatases are critical for the catabolism of distinct substrates. Mice deficient in both LAP and TRAP, however, display a severe phenotype that is more than a mere addition of clinical signs seen in LAP- and TRAP-deficient mice from which the LAP/TRAP-deficient mice were generated by mating,4 e.g. cells that appear normal in mice with a deficiency of either LAP or TRAP, such as Kupffer cells of liver, show excessive lysosomal storage. This strongly suggests that for a number of substrates, LAP and TRAP can substitute for each other. Storage of such substrates is therefore only observed if both lysosomal phosphatases are deficient.


FOOTNOTES

*   This work was supported in part by Deutsche Forschungsgemeinschaft Grant Pe 310/6-1 and by the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed. Tel.: 49-551-395932; Fax: 49-551-395979; E-mail: saftig{at}uni-bc2.gwdg.de.
**   Present address: Medizinische Molekularbiologie, Innere Medizin I, Universitätsklinik Freiburg, 79106 Freiburg, Germany.
Dagger Dagger    Present address: Hoffmann-La Roche, 4058 Basel, Switzerland.
§§   Supported by a Federation of European Biochemical Societies short-term fellowship. Present address: Nencki Inst. of Experimental Biology, Warsawa 02-093, Poland.
1   The abbreviations used are: LAP, lysosomal acid phosphatase; TRAP, tartrate-resistant type 5 acid phosphatase; kbp, kilobase pair(s); ES, embryonic stem; GSA, G. simplicifolia agglutinin; RCA, R. communis agglutinin.
2   R. Pohlmann, unpublished data.
3   W. Löscher and W. Danysz, personal communication.
4   A. Suter and P. Saftig, unpublished data.

ACKNOWLEDGEMENTS

We thank N. Hartelt, H. Boetcher, A. Wolff, and B. Rühlke for excellent technical assistance and W. Schmahl and C. Rahner for performing analyses of various tissues of LAP-deficient animals. We are grateful to W. Danysz (Merck, Frankfurt, Germany) for performing behavioral studies and to W. Löscher for examining epileptic seizures in LAP-deficient mice. We thank R. Kunze for two-dimensional lipid analysis of LAP-deficient fibroblasts.


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