Progressive Segregation of Unmyelinated Axons in Peripheral Nerves, Myelin Alterations in the CNS,...

Progressive Segregation of Unmyelinated Axons inPeripheral Nerves, Myelin Alterations in the CNS, and

Cyst Formation in the Kidneys of Myelin andLymphocyte Protein-Overexpressing Mice

Marcus Frank, *Suzana Atanasoski, *Sara Sancho, *Josef P. Magyar, †Thomas Ru¨licke,Martin E. Schwab, and *Ueli Suter

Brain Research Institute, Department of Neuromorphology, University of Zu¨rich and Swiss Federal Institute of Technology;* Institute of Cell Biology, Department of Biology, Swiss Federal Institute of Technology, ETH-Ho¨nggerberg; and

†Central Biological Laboratory, University Hospital of Zu¨rich, Zurich, Switzerland

Abstract: Myelin and lymphocyte protein (MAL) is a pu-tative tetraspan proteolipid that is highly expressed bySchwann cells and oligodendrocytes as a component ofcompact myelin. Outside of the nervous system, MAL isfound in apical membranes of epithelial cells, mainly inthe kidney and stomach. Because MAL is associated withglycosphingolipids, it is thought to be involved in theorganization, transport, and maintenance of glycosphin-golipid-enriched membrane microdomains. In this report,we describe the generation and analysis of transgenicmice with increased MAL gene dosage. Immunohisto-chemical analysis revealed that the localization of MALoverexpression in the transgenic animals correspondedclosely to the MAL expression pattern observed in wild-type animals, indicating correct spatial regulation of thetransgene. Phenotypically, MAL overexpression led toprogressive dissociation of unmyelinated axons frombundles in the PNS, a tendency to hypomyelination andaberrant myelin formation in the CNS, and the formationof large cysts in the tubular region of the kidney. Thus,increased expression of MAL appears to be deleteriousto membranous structures in the affected tissues, indi-cating a requirement for tight control of endogenousMAL expression in Schwann cells, oligodendrocytes,and kidney epithelial cells. Key Words: Schwann cell—Oligodendrocyte—Membrane microdomain—Protein trans-port.J. Neurochem. 75, 1927–1939 (2000).

Large amounts of membrane constituents have to besynthesized by oligodendrocytes and Schwann cellswithin a few days during myelinogenesis, a process thatleads to a several hundredfold increase of the membranesurface area in these cells (reviewed by Morell, 1984;Pfeiffer et al., 1993). The generation of myelin proteinsand lipids and the wrapping of the membrane processesaround the axons require the coordinated expression ofmyelin genes. Malfunctions of the products of some of

these genes are known to cause severe myelin disordersin the CNS and PNS (reviewed by Snipes and Suter,1995; Scherer, 1997).

Several of the major proteins of the myelin sheathhave been cloned, and their functions have been studiedin transgenic animals overexpressing, misexpressing, orlacking these proteins (reviewed by Werner et al., 1998).Absence of the proteolipid protein (PLP) and its splicevariant DM20, which account for up to 50% of the totalmyelin protein in the CNS, is largely compatible with theinitial assembly of compact myelin sheaths but is fol-lowed by widespread axon swellings and degeneration(Boison and Stoffel, 1994; Klugmann et al., 1997; Grif-fiths et al., 1998). Mutations in PLP affect its intracellu-lar transport, resulting in oligodendrocyte death (Gowand Lazzarini, 1996; Gow et al., 1998). Compaction ofthe peripheral myelin sheath is impaired in null mutantmice of the most abundant peripheral myelin protein,protein 0 (P0) (Giese et al., 1992), and mutations affect-ing P0 are associated with hereditary neuropathies inhumans (reviewed by Warner et al., 1996). Similarly,absence, overexpression, or mutations of peripheral my-elin protein 22 (PMP22) lead to myelin defects in hu-mans and rodents (Adlkofer et al., 1995; Magyar et al.,1996; Sereda et al., 1996; reviewed by Naef and Suter,

Received April 5, 2000; revised manuscript received June 19, 2000;accepted June 30, 2000.

Address correspondence and reprint requests to Dr. U. Suter atInstitute of Cell Biology, Department of Biology, Swiss Federal Insti-tute of Technology, ETH-Ho¨nggerberg, CH-8093 Zu¨rich, Switzerland.E-mail: [email protected]

Drs. M. Frank, S. Atanasoski, and S. Sancho contributed equally tothis work.

Abbreviations used:CGT, galactose:ceramide galactosyltransferase;MAL, myelin and lymphocyte protein; NPH, juvenile nephronophtisis;P0, protein 0; PBS, phosphate-buffered saline; PLP, proteolipid pro-tein; PMP22, peripheral myelin protein.

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Journal of NeurochemistryLippincott Williams & Wilkins, Inc., Philadelphia© 2000 International Society for Neurochemistry

1998). Furthermore, connexin32, which is confined tononcompacted myelin domains, is involved in X-linkedhereditary motor and sensory neuropathies (Bergoffenet al., 1993; Anzini et al., 1997). The function of specificmyelin lipids has also been investigated in transgenicmice (reviewed by Coetzee et al., 1996, 1998; Stoffel andBosio, 1997). In particular, studies of mice lacking UDP-galactose:ceramide galactosyltransferase (UDP-CGT),the key enzyme of the glycosphingolipid pathway,showed that glycosphingolipids are essential componentsfor the proper functioning of the myelin sheath.

The highly hydrophobic myelin and lymphocyte pro-tein (MAL) has recently been characterized as an addi-tional myelin component (Kim et al., 1995; Schaeren-Wiemers et al., 1995a; Frank et al., 1998). Human MALhad previously been cloned from T-cell lines (Alonsoand Weissman, 1987; Rancano et al., 1994), and canineMAL was isolated from the Madin–Darby canine kidneyepithelial cell line as a constituent of apical transportvesicles (Zacchetti et al., 1995; Millan et al., 1997). Wehave demonstrated that MAL is also associated withglycosphingolipids in myelin (Frank et al., 1998). Out-side the nervous system, MAL is expressed in the apicalmembranes of epithelial cells in kidney and stomach,which both show a glycosphingolipid composition sim-ilar to myelin (Frank et al., 1998). These findings suggestthat MAL may be involved in the formation, transport,and/or maintenance of specific glycosphingolipid-en-riched membrane domains, thus contributing to essentialproperties of specialized myelin and epithelial mem-branes, such as presenting barriers to diffusion of smallmolecules (Zacchetti et al., 1995; Frank et al., 1998;Cheong et al., 1999; Puertollano et al., 1999).

To gain more insight into the functional roles of MALin myelin and epithelial membranes, we have generatedtransgenic mice overexpressing MAL under its own reg-ulatory elements. We show that MAL overexpressionresults in a peculiar segregation of unmyelinated axonsfrom bundles in peripheral nerves, a tendency to mildhypomyelination and myelin alterations in the CNS, andsevere cyst formation in the kidney. These findings areconsistent with the hypothesis that correct levels of MALexpression are involved in the formation and mainte-nance of myelin and kidney epithelial membranes invivo.

MATERIALS AND METHODS

Generation of transgenic miceThe; 34-kb insert of cosmid pTCF-MAL2.1 containing the

mal gene flanked by 8 kb of upstream nontranscribed region(Magyar et al., 1997) was released byNruI restriction endonu-clease digestion and isolated using the Biotrap electroelutor(Schleicher & Schuell, Dassel, Germany). DNA was desaltedand equilibrated in injection buffer [5 mM Tris-HCl (pH 7.5),5 mM NaCl, and 0.1 mM EDTA] using a Centricon 30 con-centrator (Amicon, Beverly, MA, U.S.A.). After filtration witha prewashed Ultrafree-MC filter unit (pore size, 0.22mm;Millipore, Bedford, MA, U.S.A.), DNA was diluted to 2 ng/mland microinjected into the pronucleus of zygotes from super-

ovulated B6C3F1 female mice. Surviving embryos were reim-planted into pseudopregnant foster mothers, and founder ani-mals were born.

Southern blot and PCR analysisTo determine the transgene copy number of the founder

animals, a 1.7-kbKpnI–EcoRI fragment corresponding to basepairs 172–1,897 of the mouse MAL cDNA (EMBLGeneBankaccession no. Y07626) was used as a probe for Southern blotanalysis. Signal intensity values were determined using a com-puter-assisted image analysis system (Fluor-S MAX MultIm-ager; Bio-Rad). Transgenic progeny was identified by PCRanalysis of mouse tail genomic DNA using the pTCFupperprimer CCTCAACCTACTACTGG (Magyar et al., 1997) andthemal gene internal primer GTCAGGGTTTCTATTCC (Fig.1A). Conditions for hot-start PCR reactions were a 4-min initialdenaturation, followed by 35 cycles of 94°C for 45 s, 55°C for1 min, and 72°C for 1 min using a thermal cycler (GeneAmpPCR System 9600; Perkin-Elmer).

RNA preparation and analysisOne-year-old MAL-transgenic mice and wild-type litter-

mates were killed, and dissected tissues were frozen in liquidnitrogen, pulverized with a frozen mortar and pestle, and sub-sequently homogenized in guanidinium thiocyanate buffer. Ly-sates were centrifuged over cushions of 5.7M CsCl2 at 32,000rpm in a Beckman SW40 rotor for 24 h at 25°C. Pellets wereresuspended in 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (pH8.0), extracted once with equal parts of phenol and chloroformand once with chloroform and butanol (80:20 vol/vol), ethanol-precipitated, and stored in diethylpyrocarbonate-treated waterat 280°C. Northern blot analysis was performed with 10mg oftotal RNA per lane on 1% agarose gels containing 16% form-aldehyde and blotted overnight onto nitrocellulose filters (Op-titran BA-S 83 Reinforced NC; Schleicher & Schuell). TheRNA was cross-linked using a UV Stratalinker (Stratagene, SanDiego, CA, U.S.A.). A 2.2-kbEcoRI fragment of the mouseMAL cDNA (EMBLGenBank accession no. Y07626) was la-beled by random primer synthesis, and hybridizations wereperformed using 5–103 105 cpm/ml solution.

Animals and tissuesMice of various ages were deeply anesthetized with pento-

barbital (Nembutal; Abbott, North Chicago, IL, U.S.A.) andtranscardially perfused with a fixative containing 4% parafor-maldehyde in pH 7.5 phosphate buffer. Brain, spinal cord,kidney, stomach, and peripheral nerves were removed, post-fixed in the same fixative overnight, immersed in 30% sucrosefor cryoprotection, embedded in Tissue Tek (Sakura, Torrance,CA, U.S.A.), and frozen in liquid isopentane at240°C. Forelectron microscopy, optic nerves and parts of the spinal cordand peripheral nerves were postfixed in phosphate buffer con-taining 2.5% glutaraldehyde. In addition, five animals 2–3months old and two animals;1 year old were perfused with4% paraformaldehyde and 2.5% glutaraldehyde in 0.1M caco-dylate buffer. Tissues were removed, postfixed in the samefixative overnight at 4°C, and processed for electron micros-copy or paraffin embedding.

AntibodiesRabbit anti-rat MAL antibodies were described by Frank

et al. (1998). Rabbit anti-mouse MAL antibodies were gener-ated at Research Genetics (Huntsville, AL, U.S.A.) using the13-mer peptide MFDGFTYKHYHEN corresponding to aminoacids 114–126 of mouse MAL as immunogen and the multipleantigenic peptide method for immunization (Posnett et al.,

J. Neurochem., Vol. 75, No. 5, 2000

1928 M. FRANK ET AL.

1988). A Sepharose 4B column with peptides linked via aDKDK spacer (Research Genetics) was used to affinity-purifythe antisera according to standard protocols (Harlow and Lane,1988). After a salt wash with 500 mM NaCl, specific antibodieswere obtained in acidic elution steps as described (Frank et al.,1998).

Immunocytochemistry and histologyCryosections 15–20mm thick were cut for immunochemis-

try and stained as described previously (Frank et al., 1998). Inbrief, myelinated tissues were permeabilized with ice-cold 95%ethanol and 5% acetic acid for 25 min, rehydrated in phosphate-buffered saline (PBS), and blocked for 1 h with PBS containing0.1% coldwater fish gelatin (Aurion, Wageningen, The Neth-erlands), 2.5% normal goat serum, and 0.05% saponin (Sigma,Buchs, Switzerland). All antibody incubation steps were per-formed in this buffer, whereas PBS was used for washing steps.For sections of unmyelinated nerves like the sympathetic trunk,the permeabilization step was omitted. Similarly, for kidneyand stomach, only mild permeabilization was required. PBScontaining 0.1% saponin and 5% normal goat serum was usedfor blocking, and the antibodies were diluted in PBS containing5% normal goat serum. After overnight incubation with anti-MAL antibodies (5mg/ml) at 4°C, signals were detected withbiotin-labeled secondary antibodies and the ABC kit (Vector,Burlingame, CA, U.S.A.) using diaminobenzidine as a chro-mogen. For plain histology, 3-mm-thick paraffin sections werestained with hematoxylin/eosin. Sections were viewed and pho-tographed under a Zeiss axiophot microscope.

Electron microscopyTissues were postfixed for 2 h with osmium tetroxide, de-

hydrated in a graded ethanol series and propylene oxide, andembedded in Araldite (Serva, Heidelberg, Germany). Thin sec-tions (0.5 mm) were cut and stained with Richardson blue.Ultrathin sections of 50–70 nm were collected on pioloform-coated nickel grids and analyzed in a Zeiss EM 902 electron

microscope operated at 50 kV. The number of unmyelinatedaxons was quantified blindly on cross-sections of optic nervesfrom wild-type and transgenic animals (n5 5). Starting at theedge of the nerve, random photographs were taken at a mag-nification of 37,000 from coded samples using the stepwisemotor positioning in thex- and y-axes of the Zeiss EM 902apparatus. Unmyelinated axons per micrograph (correspondingto an area of 125mm2) were counted on glossy prints (n5 8–11) at a final magnification of314,000. Photographstaken at the edges of the nerves were omitted for countingbecause the number of unmyelinated axons varied greatly ow-ing to different composition of the glia limitans. Mean valuesfor the number of unmyelinated axons were calculated for theindividual animals. The mean numbers observed in transgenicmice were analyzed with Student’st test and the U test (Mann–Whitney/Wilcoxon rank test) for significant differences in com-parison with the wild-type (one-side test paradigm). Five cross-sections 1mm thick, stained with alkaline toluidine blue, wereused to count nuclei in the saphenous nerves of 2-, 3-, 5-, and12-month-old transgenic and wild-type mice. All nuclei presentinside the fascicle, with the exception of nuclei related to bloodvessels, were counted. Ultrathin sections of peripheral nerveswere contrasted with uranyl acetate and lead citrate and exam-ined with a JEOL model 100C electron microscope.

RESULTS

Generation of MAL-transgenic miceTo analyze the effect of elevatedmal gene dosage in

MAL-expressing tissues, we generated transgenic miceusing the previously described cosmid clone pTCF-MAL2.1 containing themalgene flanked by;8 kb of itsupstream nontranscribed region (Magyar et al., 1997).The 34.5-kbNruI insert including ;0.2 kb of vectorsequences on either end (Fig. 1A) was microinjected into

FIG. 1. Generation of MAL-transgenicmice and northern blot analysis. A: Struc-ture of the genomic MAL cosmid clonepTCF-MAL2.1 (Magyar et al., 1997). Thefour exons are indicated by solid boxes,the 39-untranslated region is shown by ahatched box, and cosmid-derived se-quences are represented by open boxes.The 34.5-kb NruI (N) fragment of pTCF-MAL2.1 including ;8 kb of the 59-flankingregion of mal was purified and microin-jected into fertilized oocytes. K, KpnI; E,EcoRI. The KpnI–EcoRI fragment of themouse MAL cDNA (EMBLGenBank acces-sion no. Y07626) was used as a probe forSouthern blot analysis. A mal internalprimer in combination with a primer de-rived from cosmid sequences (see Magyaret al., 1997) was used for identification ofmutant genotype by PCR analysis. B: Ex-pression of MAL mRNA in 1-year-old wild-type (wt) and MAL-transgenic (TgN) sciaticnerves (lanes 1 and 2), brain (lanes 3 and4), liver (lanes 5 and 6), stomach (lanes 7and 8), and kidney (lanes 9 and 10) asanalyzed by northern blotting. Owing tothe vastly different expression levels, var-ious exposure times were required: lanes 1 and 2, 9 h; lanes 3–7, overnight; lane 8, 30 min; and lanes 9 and 10, 6 h. Equal loading wascontrolled by the main rRNA species (18S and 28S) visualized by ethidium bromide staining.


1929MAL-TRANSGENIC MICE

fertilized mouse oocytes of the B6C3F1 strain. Founderanimals were identified by Southern blot analysis ofEcoRI-digested mouse genomic DNA using aKpnI–EcoRI cDNA probe (Fig. 1A). Two founder animals withmal gene copy numbers of approximately five and 15compared with wild-type (data not shown) were bredwith wild-type B6C3F1 mice, and the offspring wereused for further analysis. Transgenic progeny were iden-tified by PCR using a cosmid-specific oligonucleotide incombination with a MAL internal primer (Fig. 1A),yielding a 350-bp transgene-specific PCR fragment (datanot shown). Unless indicated otherwise, the transgenicline with the highermal gene copy number was usedthroughout this study.

MAL-transgenic mice overexpress MAL in a tissue-and cell type-specific manner

Endogenous MAL is highly expressed in the PNS,CNS, kidney, and stomach, whereas no detectable ex-pression was observed in the liver (Schaeren-Wiemerset al., 1995b; Magyar et al., 1997). Thus, we comparedthe levels of MAL transcripts in these tissues of 1-year-old wild-type and MAL-transgenic mice by northern blotanalysis. Strongly elevated mRNA expression was foundin sciatic nerves and the stomach of MAL-transgenicmice, together with moderate increases in the kidney,whereas no obvious differences from wild-type micewere observed in the brain. As in wild-type animals, noMAL expression was detected in liver of MAL-trans-genic mice (Fig. 1B).

MAL protein expression in 2- and 12-month-oldMAL-transgenic animals and wild-type littermates wasexamined by immunocytochemistry in the spinal cord, inperipheral nerves, including sciatic nerve, femoralisnerve, spinal roots, and sympathetic trunk, and in kidneyand stomach. Weak MAL overexpression was found inmyelinated areas of the spinal cord of the transgenicmice (Fig. 2A and B), whereas more pronounced over-expression was revealed in the dorsal and ventral roots(Fig. 2A and B). Consistent with the latter observation, asimilar degree of overexpression was found in PNS my-elin of the femoralis and sciatic nerve, which consistmainly of myelinated fibers (Fig. 2C and D). It is inter-esting that the sympathetic trunk, a fiber tract that con-tains predominantly unmyelinated axons grouped in bun-dles and ensheathed by nonmyelinating Schwann cells,showed also significant MAL overexpression (Fig. 2Eand F). These findings are in agreement with our recentfinding that MAL is expressed by both myelinating andnonmyelinating Schwann cells (Frank et al., 1999).

Analogous to previous findings in the rat (Frank et al.,1998), MAL was strongly expressed at apical mem-branes of cells of the distal kidney tubuli and in cells ofthe glandular stomach epithelium in the wild-typemouse. Strong cell type-specific MAL overexpressionwas seen in transgenic mice in both organs (Fig. 3). Intransgenic animals, MAL immunoreactivity was con-fined to identical segments of nephrons as in wild-typemice. Renal histopathological changes, which became

evident already at the age of 2 months, manifested bysevere cyst formation, closely correlated with MALoverexpression (Fig. 3A and B). In the stomach, MALexpression was restricted to cells of the glandular epi-thelium in both transgenic mice and wild-type littermatesbut was much stronger in the transgenic animals (Fig. 3Cand D).

In the transgenic line with the lowmal transgene copynumber, MAL overexpression was detectable immuno-cytochemically in kidney and stomach when comparedwith wild-type littermates. Mild tubular enlargementswere present in the kidneys of one animal at 1 year ofage, but a potential incidence of spontaneous diseasecannot be discounted (data not shown). Similar to theresults obtained in animals with the highermal genecopy number, strongest MAL immunoreactivity wasfound in the stomach and in the kidneys, but the stainingwas less pronounced (data not shown). Based on thesedata, the analysis of this line of mice has been discon-tinued.

CNS myelin alterationsThe optic nerve was systematically examined in

2-month- and 1-year-old animals to reveal potential mor-phological changes in the MAL-transgenic animals. Thegross morphology of the nerve and the cytoarchitectureof the glial cells were not altered, but various changes atthe subcellular level were revealed. In 2–3-month-oldmice, an unusually large number of unmyelinated axonswas observed, suggesting hypomyelination (Fig. 4B andC, asterisks). Quantification on electron micrographs in-dicated that the number of unmyelinated axons was mod-erately increased in the MAL-transgenic animals (Fig.4E). The mean6 SEM value for unmyelinated axons permicrograph was;50% higher in transgenic mice (n5 5;30 6 4.2 axons per micrograph) than in the wild-type (n5 5; 186 4.2 axons per micrograph). Although consid-erable variation between individual animals was evident(Fig. 4E), the increase in the number of unmyelinatedaxons was significant atp , 0.05 (one side), in botht test( p 5 0.046) and U test. Similarly, mild hypomyelinationwas indicated by the seeming predominance of small andmidsized myelinated fibers at some places in the opticnerve of transgenic animals compared with wild-typeanimals. However, normal, large-caliber axons were alsofound in the transgenic animals, and the general variabil-ity of axon sizes in wild-type and transgenic optic nervesprecluded further statistical analysis.

In addition, abnormal myelin formations were ob-served in the MAL-transgenic animals (Fig. 4B–D). Atplaces the myelin sheath seemed too large in relation tothe axon diameter, often resulting in the formation of agap between the axon membrane and the inner cytoplas-mic loop of the sheath (Fig. 4B and C, arrows). Aberrantmyelin formations were also associated with abnormal-ities of the inner mesaxon. Here, fixation artifacts andmisformation of myelin membranes occurred more fre-quently in the transgenic animals (Fig. 4C, arrows).Unusual myelin depositions in large whirls or around



seemingly empty spaces was also observed, but the gen-esis of these structures and their relationship to potentialaxonal degeneration remain unclear (Fig. 4D). The sameobservations were made in 1-year-old transgenic miceand seemed to have progressed with age. However, po-tential age-related changes like spontaneous degenera-tion of retinal ganglion cell axons precluded a conclusiveevaluation with respect to the causative effects of MALoverexpression (data not shown).

Progressive segregation of unmyelinated axons inMAL-transgenic mice

Peripheral nerves from 2–3- and 12-month-old MAL-transgenic mice and wild-type littermates were compared

by electron microscopy. No overt alterations were ob-served in the cytoarchitecture of myelinated fibers or inSchwann cell myelin in transgenic mice. In contrast,MAL-transgenic nonmyelinating Schwann cells showeda striking phenotype. As a rule, unmyelinated axons inwild-type nerves were ensheathed together in bundles ofvarious sizes by individual Schwann cells (Figs. 5A andC and 6A and C). The number of axons present in eachbundle varied widely, but unmyelinated fibers usuallycontained between five and.20 axons. Individualiza-tions of single unmyelinated axons were very rarelyobserved. Few collagen fibers were interspersed amongthe unmyelinated axon–Schwann cell units. In contrast,

FIG. 2. Expression of MAL in the nervous system.Wild-type (wt) and transgenic (tg) mice were ana-lyzed by immunocytochemistry for MAL expres-sion on sections of 2-month-old (A and B) spinalcord, (C and D) sciatic nerve, and (E and F) sym-pathetic trunk. Compared with wt (A), MAL immu-noreactivity is slightly increased in the spinal cordof the tg mouse (B). In cross-sections of the sciaticnerve, MAL is localized in the myelin sheaths, re-vealing the typical ring-like immunoreactive struc-tures around the axons (C and D). Increased stain-ing is found in the tg animal (D) compared with wt(C). On cross-sections of the sympathetic trunk (Eand F), diffuse MAL immunoreactivity is observedin the neuropil; stronger staining is present in the tganimal (F) compared with wt (E). Cell nuclei andblood vessels remain unstained; the nonspecificreaction of erythrocytes containing endogenousperoxidase is the same in the nerves of both ani-mals. Bars 5 120 mm in A and B, 18 mm in C andD, and 60 mm in E and F.



all transgenic nerves examined showed a progressivesegregation and individualization of unmyelinated axons.Such a process was already evident at 2 months of age,especially in the sympathetic trunk where many unmy-elinated axons appeared completely detached from eachother (Fig. 6B). Although individualization of unmyeli-nated axons was less apparent in the somatic nerves ofMAL-transgenic animals at 2 months of age (Fig. 5B),unmyelinated fibers had a less compacted structure andbundles contained fewer axons compared with wild-typenerves (Fig. 5A). In older animals, axonal segregationhad proceeded to become evident at the light microscopylevel. When compared with the saphenous nerve ofyoung animals (Fig. 5a and b) or 1-year-old littermates(Fig. 5c), the saphenous nerves of transgenic miceshowed increased endoneurial space between groups ofmyelinated fibers (Fig. 5d). Ultrastructural examinationrevealed that the increased endoneurial space observedby light microscopy was filled with segregated unmyeli-nated axons and abundant collagen fibers, but no majoralterations in axon number were evident (Fig. 5D).Whether there is a minor loss of small fibers remains tobe determined. Schwann cell cytoplasmic tongues ortheir basal lamina surrounded the unmyelinated axons.Occasionally, a long Schwann cell cytoplasmic processwas seen to bridge between two segregated axons (datanot shown). Complete individualization of unmyelinatedaxons had advanced in the sympathetic trunk as well, but

collagen deposition was less prominent. Consistently,individualized axons in the sympathetic trunk were com-pletely surrounded by Schwann cell cytoplasmic pro-cesses (Fig. 6D). Similar findings were made in otherperipheral nerves, including sciatic, quadriceps, and oc-ulomotor nerves (data not shown).

One might speculate that aberrant proliferation ofnon–myelin-forming Schwann cells may contribute tothe observed phenotype. Thus, to evaluate whether in-creased Schwann cell number coincides with the pro-gressive segregation of unmyelinated axons, we countedthe total number of nuclei present in the fascicular area ofthe saphenous nerves of 2–12-month-old transgenic andcontrol mice. The number of nuclei appeared to be in-creased between 1.2- and 1.7-fold in the saphenousnerves of MAL-transgenic animals. By electron micro-scopic analysis the number and appearance of fibroblastsand endothelial cells appeared to be not significantlyaltered, and no signs of infiltrating inflammatory cellswere observed (data not shown). This indicates that theincreased number of nuclei is likely due to a highernumber of Schwann cells.

MAL overexpression in the kidney causesprogressive cyst formation in distal tubuli andatrophy of the cortex

Comparison of native kidneys from wild-type andtransgenic animals revealed severe progressive

FIG. 3. Immunocytochemical analysis of MAL expression in kidney and stomach. A and B: Strong MAL overexpression is seen in thekidney of transgenic (tg) animals at the age of 2 months versus wild-type (wt) littermates. Note the cyst formation in the kidney cortexof the tg animals (B). C and D: Massive overexpression of MAL was detected in the stomach of tg mice compared with wt. MALexpression was limited to the glandular part of the epithelium in wt and tg animals. The expression stops at the transition zone (tz) tothe nonglandular epithelium, which is devoid of MAL immunoreactivity (D). Bar 5 770 mm in A and B and 200 mm in C and D.



changes in organ size and shape (data not shown). At2 months of age, kidneys from wild-type and trans-genic mice are indistinguishable by macroscopic ex-amination. In contrast, kidneys from 1-year-old trans-genic mice had a flattened, more longitudinal shape,indicating degenerative tissue alterations. The surfaceof the organ appeared to be dotted with grain-like

structures that were absent in wild-type tissue. Lightmicroscopy revealed that these structures correspondto large cysts (Fig. 7).

Adjacent sections were either stained with cresyl vio-let to visualize the cellular structure (Fig. 7A and C) orprocessed for MAL immunocytochemistry (Fig. 7B andD). All cysts were lined by MAL immunoreactivity (Fig.

FIG. 4. Ultrastructural alterations in optic nerve shown in electron micrographs of optic nerves from 2-month-old (A) wild-type (wt) and (B–D)MAL-transgenic (tg) mice. The number of unmyelinated axons (asterisks) was increased in the tg animals (B and C; for quantification, see E).At places, malformation of the myelin sheath and aberrant myelin deposition were observed in the tg animals. Gap formation between themyelin sheath and the axon (arrows in B and C) occurred often in conjunction with malformation of the mesaxon (C, arrows). Small andmidsized myelinated fibers appeared more predominant compared with wt. Also, cytoplasmic pockets in the myelin spiral (C, arrowhead) andmyelin deposits without apparent association with axons were found (D). Such alterations were largely absent in wt mice (A), where thefrequency of unmyelinated axons was low. Bars 5 560 nm in A and B and 430 nm in C and D. The numbers of unmyelinated axons werequantified on electron micrographs from optic nerves of tg and wt mice (see Materials and Methods). E: Numbers of unmyelinated axons permicrograph (125 mm2) are plotted for the individual wt and tg animals in a histogram. Data are mean values (n 5 5). The mean numbers ofunmyelinated axons in the tg animals are significantly increased (p , 0.05, one side) compared with wt in t test and U test (Mann–Whitney/Wilcoxon rank test) analysis. The total mean value of unmyelinated axons per micrograph was increased in the tg mice (30 6 4.2 axons permicrograph; mean 6 SEM) compared with wt (18 6 4.2 axons per micrograph).



FIG. 5. Morphological alterations in peripheral nerves of MAL-transgenic (MAL-Tg) mice shown in semithin section (a–d) and electronmicrographs (A–D) of the saphenous nerve of 2-month-old (a, b, A, B) and 1-year-old (c, d, C, D) wild-type (a, c, A, C) and MAL-Tg (b,d, B, D) mice. The gross morphology of saphenous nerves of 2-month-old wild-type (a), 2-month-old MAL-Tg (b), and 12-month-oldwild-type (c) mice are indistinguishable by light microscopy. Apparently increased endoneurial space (arrowheads), interposed betweengroups of myelinated fibers, is prominent in the saphenous nerve of 12-month-old MAL-Tg mice (d). The ultrastructure of wild-typenerves (A and C) shows varying numbers of unmyelinated axons (marked by a) packed together in well-defined groups amongmyelinated fibers (marked by A; m indicates myelin). The bundles of unmyelinated fibers appear less compact in the saphenous nerveof 2-month-old MAL-Tg mice (B). Few unmyelinated axons (arrowheads) surrounded by Schwann cell cytoplasm appear detached fromthe bundles. Many unmyelinated axons (arrowheads) are completely individualized and surrounded by increased amount of collagenfibers (marked by asterisks) in the saphenous nerve of 1-year-old MAL-Tg mice (D). Bars 5 50 mm in a–d and 2 mm in A–D.


7, arrows). In 2-month-old transgenic animals, tubularenlargements and the beginning of cyst formation wereobserved in the renal cortex (Fig. 7A and B). In 1-year-old animals, the frequency and diameter of the cysts wereremarkably increased (Fig. 7C and D). Histological ex-amination showed a dramatic atrophy and degenerationof the kidney cortex tissue due to extensions of the cyststructures, interstitial fibrosis, and inflammation (Fig.7C). A close correlation was observed between MALexpression and cyst development. The affected tubularsegments express the highest levels of MAL, both inwild-type and in transgenic mice, as judged by immuno-cytochemistry. In addition, enlarged tubuli were alsoseen in the kidney medulla. Typically, the luminal cystmembranes were outlined by strong MAL immunoreac-tivity (Fig. 7D). Closer analysis revealed that the cystslikely developed from the distal convoluted and connect-ing tubuli because the cytology of the cells lining thecysts resembled the normal cellular morphology of thesestructures, even in 1-year-old animals (Fig. 7E and F). Inaddition, normal-appearing distal convoluted tubuli wererarely identified in 1-year-old transgenic mice, whereasmore proximal segments of nephrons located in the renal

cortex such as the proximal convoluted tubuli seemedundamaged. Often, cells lining the cysts were piled up,and thus their apical sides protruded into the cyst lumen(Fig. 7G, arrow). No such alterations were observed inwild-type mice at any age (Fig. 7E).

MAL overexpression may affect the structure of theglandular gastric epithelium

The strongest MAL overexpression at the transcriptand protein level was found in the glandular gastricepithelium. Although initial observations indicated thatthe gastric mucosa of two 1-year-old transgenic animalsmay be thicker and more folded compared with wild-type animals (data not shown), the large normal variabil-ity of these structures precluded a definitive assessment.Detailed analysis with large numbers of animals will berequired to determine the effects of the robust overex-pression of MAL in this tissue.

DISCUSSION

Increasing the gene dosage ofmal controlled by itsown regulatory elements in transgenic mice resulted in

FIG. 6. Morphology of sympathetic trunk shown in electron micrographs of the sympathetic trunk of (A and B) 2-month- and (C and D)1-year-old (A and C) wild-type and (B and D) MAL-transgenic (MAL-Tg) mice. In the wild-type nerves (A and C), numerous unmyelinatedaxons (a) are held together in compact, well-defined bundles by Schwann cell cytoplasmic processes. In 2-month-old MAL-Tg mice (B),unmyelinated fiber bundles contain few axons (a). Some unmyelinated axons (arrowheads) surrounded by a Schwann cell cytoplasmictongue are completely singled out from neighboring axons. The number of individualized unmyelinated axons (arrowheads) hasincreased in 12-month-old MAL-Tg mice (D). Few myelinated axons (A) are scattered among the unmyelinated nerve fibers in all fournerves shown. m, myelin sheath; ScN, nuclei of nonmyelinating Schwann cells. Bar 5 2 mm.



increased MAL levels in organs and tissues that expressMAL also in the wild-type animal. These include thekidney, the stomach, and the nervous system. Whereasperipheral myelin appeared normal, a striking segrega-tion of nonmyelinated axons became pronounced withincreasing age. In the CNS, signs of mild hypomyelina-tion with an increased number of unmyelinated axonsand focal myelin alterations were observed. Severe cyst

formation and cortex atrophy were found in the kidney ofthe transgenic animals.

A morphological phenotype is caused by MALoverexpression

We have observed these severe anatomical alterationsin the highmal gene copy number transgenic mice only,leaving the formal possibility that a specific insertion

FIG. 7. Kidney histology in MAL-transgenic (tg) mice. The gross histology of kidneys of MAL-tg mice was analyzed by cresyl violetstaining (A and C) and compared with MAL immunoreactivity (B and D) on adjacent sections. Small cysts were apparent in the distaltubuli of the kidney cortex (cx) at 2 months of age (A, arrows). These cysts are lined with strong MAL immunoreactivity (B, arrows). Inaddition, weaker MAL staining is seen at other distal tubuli in the kidney medulla (md). In 1-year-old animals, cysts were considerablylarger in size, closely corresponding to the pattern of MAL immunoreactivity (C and D, arrows). Tubular enlargements were also presentin the kidney medulla (arrowheads, C and D). Alterations in cytoarchitecture were examined on hematoxylin/eosin-stained paraffinsections of kidneys of (F and G) MAL-tg and (E) wild-type mice at 1 year of age. Normal renal cortex (E) shows distal convoluted tubuli(asterisks) next to a glomerulus (g). Note the apical distribution of the nuclei in the distal tubuli. The morphology and appearance of thecells lining the wall of the cysts in the kidney of MAL-tg mice (F and G, asterisk) are reminiscent of the epithelial cells of the distalconvoluted tubuli in the wild-type (E, asterisk). Often, cells in the cyst walls are piled up with pseudostratification of the nuclei, and theirapical membrane protrudes into the cyst lumen (G, arrowheads). Bars 5 340 mm in A–D and 20 mm in E–G.



artifact may contribute to the phenotype. However, forthe following reasons we believe that the observed mor-phological phenotype is consistent with a causative ef-fect of MAL overexpression: (a) MAL overexpressionwas confined to the tissues and cells that also expressMAL endogenously. (b) Attempts to generate additionalmouse strains with similar or higher copy numbers of themal transgene were not successful, i.e., they were asso-ciated with premature death of founder animals, andmice with lowermal gene copy numbers did not yieldclear anatomical defects. Thus, overexpression of MALmay only be compatible with life and causing anatomicalalterations within a small expression window. (c) Arecent report on the effects of overexpression of MAL inMadin–Darby canine kidney cells in vitro (Cheong et al.,1999) appears consistent with the observations in ourMAL-transgenic mice: MAL overexpression increasedthe apical surface of the Madin–Darby canine kidneycells and caused morphological changes of the apical cellmembranes. We have noted a similar protruding of theapical membranes of cells in the kidney tubuli of theMAL-transgenic mice. In vivo, supernumerary mem-brane production caused by MAL overexpression maypromote the massive cyst formation and may contributeto the loosened myelin sheaths and myelin malforma-tions in the CNS, as well as to the segregation of axonsby nonmyelinating Schwann cells. Taken together, thesearguments suggest that a potential insertion artifact ap-pears rather unlikely as the cause for the phenotype wehave observed.

Transgenic MAL expression is tissue- and celltype-specific

Recent analysis of themal gene and its promoterrevealed recognition sites for the transcription factor Sp1and othercis-acting regulatory elements within the first600 bp of the 59-upstream flanking region. These ele-ments are sufficient to drive reporter gene expression invitro independent of the cell type used (Tugores et al.,1997). Approximately 8 kb of themal59-upstream flank-ing region was present in the transgene used in this study,resulting in tissue- and cell-specific MAL overexpres-sion. These results are consistent with the hypothesis thatregulatory elements required for tissue-specific MALgene expression must be present far upstream of the corepromoter (Tugores et al., 1997). However, the quantita-tive expression of themal transgene differs among thetissues examined. High overexpression was observed inSchwann cells and the stomach epithelium, moderatelevels in the kidney, and low levels in oligodendrocytes.Thus, this MAL transgene is likely to lack regulatoryelements that are required for correct expression levels insome of the endogenously MAL-expressing tissues.

Intrinsic control of MAL expression levelsOur data suggest that close control of MAL protein

expression in glial cells and in the kidney appears to becrucial because increasing MAL expression leads to se-vere alterations in these organs and tissues. Thus, one

might speculate that similar to other myelin genes, in-cluding PLP and PMP22, the MAL gene might be sen-sitive to increased gene dosage (reviewed by Suter andSnipes, 1995; Werner et al., 1998). However, the effectsof overexpression of MAL as a component of compactperipheral myelin is distinctly different from overexpres-sion of PMP22, which is also localized to compact do-mains of PNS myelin (Magyar et al., 1996; Sereda et al.,1996; Sancho et al., 1999). Whereas PMP22 overexpres-sion leads to severe myelin defects and secondary axonalchanges, MAL overexpression in the PNS affects pre-dominantly nonmyelinating Schwann cells with no ap-parent alterations of peripheral myelin. In the CNS, my-elin abnormalities were present but of a relatively mildtype.

Functional implicationsDuring embryonic development, MAL is expressed in

immature Schwann cells before myelination (Franket al., 1999), and resident MAL expression is seen innonmyelinating Schwann cells in the adult animal. Thus,MAL expression might be involved in the terminal tran-sition step of Schwann cell differentiation when myeli-nating and nonmyelinating cells are separated. It is in-teresting that it has been reported that this step involvesthe expression of sulfatide (Mirsky et al., 1990), a gly-cosphingolipid found to be also biochemically associatedwith MAL (Frank et al., 1998). Thus, in the embryo,MAL expression might promote the segregation of axonsby Schwann cells as a prerequisite for myelination. Con-tinuous overexpression of MAL could mimic this situa-tion in the adult, potentially explaining the observedaberrant segregation of unmyelinated axons from bun-dles. Myelination of the segregated axons may not occurat this late time, however, because crucial differentiationfactors like the transcription factors Krox20 and SCIP/Oct6 are not expressed at the required levels (Topilkoet al., 1994; Zorick et al., 1996; Arroyo et al., 1998).Alternatively, the small diameter of the axons may pre-clude them from myelination.

In the CNS, we observed a complex mixture of hypo-myelination, as suggested by an increase in the numberof unmyelinated axons in the optic nerve and focal my-elin malformations. It is interesting that similar alter-ations have been described in the CNS of mice that lackCGT and, as a consequence, cannot properly generate themajor myelin glycosphingolipids (Coetzee et al., 1996,1998). As MAL is a constituent of glycosphingolipid-rich membrane domains, it may contribute to myelintightness and stability (Frank et al., 1998). Furthermore,overexpression of MAL may severely alter the physiol-ogy of the transgenic cells because increased levels ofMAL might titrate out glycosphingolipids, thereby po-tentially mimicking CGT deficiency. In addition, giventhe phenotype observed in kidney, one might speculatethat MAL may have a function as an “adhesive pore” andthus compose both membrane adhesion and electrolytebalance, as has been suggested previously for other tet-raspan proteolipid proteins (Kitagawa et al., 1993).



In the kidney, cyst formation primarily affects thedistal convoluted and connecting tubuli, which also showthe highest MAL immunoreactivity in the transgenicanimals and in wild-type mice. MAL is expressed earlyduring rat kidney development (Frank et al., 1998), andMAL transcripts have been found to be up-regulatedduring differentiation of urothelial cells in vitro (Liebertet al., 1997). It is striking that again glycosphingolipidsare known to be involved in these processes. Renalgrowth and cell proliferation are associated with changesin glycosphingolipid levels, and altered amounts of gly-cosphingolipids were also found in polycystic kidneys ofthe mouse mutantcpk/cpk (for review, see Shayman andRadin, 1991; Shayman, 1996). Thus, elevated levels ofglycosphingolipid-associated MAL (Frank et al., 1998)may locally affect the rate of cell proliferation in theconnecting tubules. Indeed, preliminary analysis sug-gests that increased proliferation of MAL-overexpress-ing cells may contribute to this phenotype (S.A. and S.S.,unpublished data).

Recently, the locus for juvenile nephronophthisis(NPH), a hereditary polycystic kidney disease in hu-mans, has been assigned to the geneNPHP1on chromo-some 2q13, in a distance of a few centimorgans to theMAL gene locus (Alonso et al., 1988; Konrad et al.,1996; Hildebrandt et al., 1997). It is interesting that insome patients the deletions of the NPHP1 locus includethe first exon of the BENE gene (Hildebrandt et al.,1997). BENE is a member of the MAL proteolipid fam-ily (Lautner-Rieske et al., 1995; Magyar et al., 1997;Perez et al., 1997). Moreover, the progressive nature ofthe disease and some of the histopathological changesthat are observed in the kidneys of nephronophthisispatients are mirrored by the MAL-transgenic mice. De-tailed investigation of the polycystic kidney phenotype inour transgenic mouse model and progress in humangenetics will help to clarify whether MAL might be acandidate gene for some hereditary forms of nephron-ophthisis.

In conclusion, stability and maintenance of specializedmembrane domains are the key features affected inMAL-transgenic mice. Elevated levels of MAL causealterations in cellular morphology and potentially alterthe cell proliferation. The distinct progression of thephenotypes with increasing age and the close correlationbetween endogenous and exogenous MAL expressionwithin the affected tissues indicate that the observedalterations are the result of continuous MAL overexpres-sion.

Acknowledgment: This work was supported by grants ofthe Swiss National Science Foundation (to S.A. and U.S. andgrant 31-45549.95 to M.E.S.), the International Institute forResearch in Paraplegia, the Vontobel Stiftung-Zu¨rich (to U.S.),and the Deutsche Forschungsgemeinschaft. We thank Drs.Marjan van der Haar and Nicole Schaeren-Wiemers for manyfruitful discussions, and Dr. Steve Scherer for valuable exper-imental advice. We acknowledge the photographic work ofRoland Schoeb and the help of Dr. Roland Du¨rr with statisticalanalysis.

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