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Isoform-selective deficit of glycine receptors in the mouse mutant spastic
Neuron,
Vol. 8, 283-289,
February,
1992, Copyright
0 1992 by Cell Press
Isoform-Selective Deficit of Clycine in the Mouse Mutant spastic Cord-Michael Becker,*+ Volker Paola Tarroni,*§ Uta Sfrasser,* and Heinrich Betz*
Schmieden,*
*Zentrum fiir Molekulare Biologie +Neurologische UniversiGtsklinik UniversitBt Heidelberg Im Neuenheimer Feld 282 D-6900 Heidelberg *Max-Planck-lnstitut ftir Hirnforschung DeutschordenstraBe 46 D-6000 Frankfurt 71 Germany
Summary The mutant mouse spastic(spa) develops a characteristic motor disorder about 2 weeks after birth, with symptoms resembling sublethal poisoning by the glycinergic antagonist strychnine. Correspondingly, adult homozygotic mutants (spa/spa) exhibit a severe reduction of inhibitory glycine receptors in spinal cord and brain. Here we show that the spastic mutation selectively interferes with the postnatal accumulation of the adult isoform of the glytine receptor protein, whereas perinatal expression of the neonatal receptor isoform is not detectably affected. Heterologous expression in X. laevis oocytes of poly(A)+ RNA and Northern blot analysis indicate normal levels of glycine receptor al subunit transcripts in spinal cord of adult spastic mutants. Thus, the age-dependent manifestation of spastic symptoms after birth reflects a selective effect of the mutation on the developmental expression of the adult glycine receptor isoform. Introduction Mice homozygous for the recessive spastic mutation (genotype spa/spa) on chromosome 3 (Either and Lane, 1980) develop a severe motor disorder during the second postnatal week(reviewed by Becker, 1990). The syndrome is characterized by muscle rigidity, tremor, and myoclonic jerks (Chai, 1961; Chai et al., 1962) and electromyographically resem bles poisoning by subconvulsive doses of strychnine (Heller and Hallett, 1982), an antagonist of the inhibitory glycine receptor (reviewed by Betz and Becker, 1988; Langosch et al., 1990). Indeed, both glycine receptor levels determined by high affinity [3H]strychnine binding (White and Heller, 1982; White, 1985; Becker et al., 1986; Rienitz et al., 1987) and glycine-mediated chloride conductance recorded electrophysiologically(Biscoe and Duchen, 1986) are drastically reduced in spa/spa mice. However, the ligand binding properties of the glycine
5 Permanent address: Centro di Farmacologia Cellulare, Dipartimento di Farmacologia Medica, Universite di Milano, Via Vanvitelli 32, l-20129 Milano, Italy.
Receptors
receptor such as affinity for [3H]strychnine and the agonistic amino acids glycine, B-alanine, and taurine are not significantly affected by the mutation (White, 1985; Becker et al., 1986). Antigenic epitopes, subunit composition, and synaptic localization of the glycine receptor complex are likewise conserved in spinal cord of adult mutant mice (Becker et al., 1986). Glycine receptors from rodent spinal cord are composed of a and p subunits. The amino acid sequences of these polypeptides deduced from corresponding cDNAs are highly homologous and share a large, extracellular N-terminal domain followed by four hydrophobic transmembrane regions. Their predicted transmembrane structure characterizes the glycine receptor subu nits as typical members of a superfamily of genes encoding ligand-gated ion channels (reviewed by Betz, 1990). Differential expression of distinct variants of the ligand-binding a subunit (Grenningloh et al., 1987; Kuhse et al., 1990a, 199Ob, 1991; Malosio et al., 1991a) contributes to heterogeneity of the receptor protein during postnatal development (Becker et al., 1988). A neonatal isoform of the glycine receptor (GlyRN) is abundantly expressed in spinal cord tissue of newborn rats (Becker et al., 1988) and in primary cultures of fetal mouse spinal neurons (Hoch et al., 1989). CI~RN exhibits only low affinity for strychnine and contains a ligand-binding subunit (a2) of 49 kd as an integral membrane-spanning polypeptide (Becker et al., 1988; Hoch et al., 1989; Kuhse et al., 1990a, 1991). GI~RN was originally identified using monoclonal antibody (MAb) 4a, which recognizes a peptide epitope common to all glycine receptor a subunits variants known (Pfeiffer et al., 1984; Becker et al., 1988; SchrBder et al., 1991). In contrast, the adult isoform of the receptor (GI~RA) binds strychnine with high affinity. GlyRn is selectively recognized by MAb 2b, which binds to an epitope at the N-terminus of the al subunit (Schrdder et al., 1991). GI~RA is thought to be a pentamer composed of an al subunit of 48 kd and a structural p subunit of 58 kd (Langosch et al., 1990). The function of the non-ligand-binding fl polypeptide is not fully understood, as heterologous expression of glycine receptorasubunitscreatesglycine-gatedchloridechannels (Schmieden et al., 1989; Sontheimer et al., 1989; Kuhse et al., 1990a, 1991). Recent cloning experiments have revealed the existence in rodent CNS of additional a subunit variants (a3 and a4), suggesting further complexity of glycine receptors (Kuhse et al., 1990b, 1991; Y. Maulet, B. Matzenbach, and H. Betz, unpublished data). However, mRNA levels in spinal cord of these novel a subunit variants are low (Malosio et al., 1991a), indicating that they correspond to minor receptorisoforms.Thus,theantibodyand radioligand probes described above permit a reliable analysis of the major glycine receptor isoforms of rodent spinal cord, GlyRN and GlyRA (Becker et al., 1988, 1989).
NlYJr0ll 284
A E
0.80
a E
0.60
Here, we have used MAbs directed against glycine receptor a subunits to analyze the expression of glytine receptor subpopulations in the adult and developing spastic mouse mutant. Our data show that the spastic phenotype of the mutant correlates with a dramatic reduction in spinal cord levels of ClyRA, but not GlyRN.
? ‘S 0.40 l? t 0.20
Moreover, the developmental appearance of symptoms coincides with the postnatal switch from neonatal to adult glycine receptor isoforms. Our data suggest a selective deficit in the regulation of ClyRA expression, which results in the age-dependent manifestation of a neurological disease.
spastic natal (calculated)
age
[postnatal
days]
Results
0
5
10 age
C
15 [postnatal
20
adult
days]
1800,
I
1500.
age Figure
1. Postnatal
Development
[postnatal of Glycine
days] Receptor
lsoforms
(A) Total glycine receptor content of rat spinal cord (open squares) was assessed by MAb 4a immunoreactivity. ClyR,, levels (open circles) were determined by high affinity [‘HIstrychnine binding. MAb 4a immunoreactivity and pHIstrychnine binding per mg of membrane protein were normalized to values found in adult animals, in which only GlyR, is detected (Becker et al., 1988). The difference between these values yielded the fraction of low affinity antagonist-binding receptors, i.e., ClyRN (closed squares). For each age, dataarethe means + SEM of 3 membrane preparations pooled from 2 rats, each. Values for adult rats were as follows: specific MAb 4a immunoreactivity, 41.8 k 6.6 AOD per mg of protein; [3H]strychnine binding, 1.32 f 0.28 pmol per mg of protein (data from Becker et al., 1988). (B) Postnatal accumulation of MAb 4a antigen in spinal cord of control (open circles) and spa/spa mice(closed circles). Antibody binding to spinal cord membranes was determined by immunoassay. (C) Postnatal expression of ClyRn as determined by binding of MAb 2b to spinal cord membranes from control mice (open circles) and spastic homozygotes (closed circles). In (B) and (C), data represent the mean f SEM of triplicate determinations of MAb 4a and MAb 2b immunoreactivities. The
Total glycine receptor levels were determined in rodent spinal cord at different postnatal ages by immunoassay employing MAb 4a, which defines an epitope common to all glycine receptor a subunits (Schroder et al., 1991). Bycomparison with selective markers of GlyRA, e.g., MAb 2b and high affinity t3H]strychnine binding (Becker et al., 1988), relative levels of GlyR,, and GI~RN were calculated from these data (Figure IA). In rat spinal cord, the postnatal accumulation of [3H]strychnine-binding sites and of MAb 2b antigen (data not shown; see Becker et al., 1988) proceeded in parallel, thus confirming their association with GlyRA (Becker et al., 1988). At birth, GlyRN accounted for about 70% of the total glycine receptor protein present. Thereafter, GI~RA increased at the expense of GI~RN, resulting in acomplete exchange of the neonatal isoform after about 2 weeks of age (Figure IA). Glycine receptor preparations purified from adult spa/spa mice display antigenic and ligand binding properties indistinguishable from those of the receptor isolated from phenotypically normal littermates (Becker et al., 1986). Thus, adult homozygous mutants express GI~RA, though at drastically reduced levels. This prompted us to study the effect of the spastic mutation on GI~RN expression. In spinal cord of control mice, the content of MAb 4a-reactive total glycine receptor increased about 60% within 1 week after birth and, thereafter, slowly declined to levels seen at birth (Figure IB). During the second postnatal week, MAb 2b immunoreactivity sharply increased from close to background values to mature levels (Figure IC). This indicates that, in the mouse, GlyRN is replaced by GlyR* at a time corresponding to that reported above for rat spinal cord development. In spastic homozygotes, the postnatal accumulation of MAb 4a and MAb 2b immunoreactivities was dramatically altered. Although spa/spa animals possessed normal amounts of MAb 4a antigen at birth, a severe reduc-
same membrane samples were applied to both immunoassays, each containing the pooled tissue from 2 mice of the indicated age and genotype. This experiment was repeated on independently collected tissue samples with essentially the same results.
Glycine
285
Receptor
lsoforms
in the spast~
Mouse
S
C h ,Z > 'Z $ L
ad
1.0
neo
neo
ad
0.8 0.6
: .f;r
0.4
T
0.2
J
4gK
-\
48K
00
neonatal Figure 2. Clycine Receptor nine Binding in Spinal Cord and spastic Homozygotes
adult lmmunoreactivity and [-‘H]strychof Neonatal and Adult Control Mice
Specific binding of MAbs 4a and 2b and of [3H]Strychnine was normalized to values obtained with adult control mice (immunoreactivities of 68.9 * 7.6 AOD per mg of protein for MAb 4a and 22.5 f 3.8 AOD per mg of protein for MAb 2b; [‘HIstrychnine binding of 934 f 111 fmol per mg of protein). Data are means f SEM of separate membrane samples (n = 2 for neonates; n = 3 for adult mice), each containing tissue from 2 mice of the indicated age and genotype. Open bars, neonatal (postnatal day 0) and adult control mice; closed bars, spastic homozygotes. Animals are different from those employed for Figure 1. Asterisks indicate that the adult spastic micediffered from control animals at p < 0.01 by the two-tailed Student’s t test for unpaired samples.
tion occurred thereafter, resulting in low adult levels (Figure IB). MAb 2b immunoreactivity indicative of GI~RA was drastically reduced in spa/spa animals throughout postnatal life (Figure ICI). Apparently, the spastic mutation has no major effect on prenatal receptor protein accumulation, suggesting that GI~RN expression proceeds normally. Using a larger sample of spinal cords from newborn spa/spa mice, the MAb 4a antigen could be identified as GI~RN by the following criteria: first, crude memneonates did not react with MAb branes from spa/spa 2b (Figure 2; compare also Figure IC); second, high affinity [3H]strychnine binding to these membranes was low, consistent with a prevalence of GI~RN (Figure 2); finally, Western blot analysis employing MAb 4a revealed a49 kd protein (Figure 3). This corresponds to the apparent molecular weight of the ligand-binding subunit of GlyRN (Becker et al., 1988; Hoch et al., 1989). In contrast to the situation at birth, membranes mice showed a significant reducfrom adult spa/spa tion in MAb4a antigen compared with control animals (Figure 2). Western blot analysis (Figure 3) revealed a polypeptide of 48 kd, i.e., the previously described al subunit of GI~RA (Becker et al., 1988; Pfeiffer et al., 1984). This is consistent with earlier observations on purified glycine receptor preparations isolated from mice (Becker et al., 1986). In accordance adult spa/spa with the low levels of MAb 2b immunoreactivity in adult mutants, [3H]strychnine binding showed only homozygotes during postnalittle increase in spastic tal life (Figure 2). These data suggest that the spastic mutation selectively affects accumulation of GlyR,+ but not GlyRN.
Figure 3. Western Blot Analysis of Clycine natal and Adult Control and spa/spa Mice
Receptor
from
Neo-
Membrane preparations were subjected to SDS-polyacrylamide gel electrophoresis and reacted with MAb 4a. Stained bands of -46 kd (adult), ~32 kd, and ~34 kd (neonatal) most likely resulted from limited proteolysis despite the use of an extended cocktail of protease inhibitors (see also Becker et al., 1988). C, control; S, spa/spa mice. Neonatal (neo), postnatal day 0; adult (ad), &weekold mice.
Xenopus laevis oocytes injected with poly(A)+ RNA isolated from rodent spinal cord express functional glycine receptors (Schmieden et al., 1989). After injection of spinal cord poly(A)’ RNA from either adult homozygotes, glycinecontrol mice or adult spastic induced inward currents were revealed by voltageclamp recording in the majority of the injected oocytes, with half-maximal responses at 180 uM and 230 uM glycine, respectively (Figure 4A). For both doseresponse relations, Hill coefficients of 3.0 were derived. Glycine currents reversed at about -25 mV, i.e., the equilibrium potential of chloride (data not shown). Variations in maximal current between different Xenopus oocytes observed with the same batch of poly(A)‘RNA, however, precluded a precisequantitation of glycine receptor transcript levels. The glycine responses obtained with both control and spa/spa poly(A)‘RNA were sensitive to strychnine, with a halfmaximal inhibition at 22 nM and 12 nM, respectively (Figure 4B). These data confirm the notion that the propertiesof GlyR~are notaltered bythespastic mutation (Becker et al., 1986). mutation alters To investigate whether the spastic the levels of glycine receptor al subunit transcripts in adult animals, we performed a Northern analysis of spinal cord poly(A)+ RNA isolated from homozygotic spastic and wild-type animals. As shown in Figure 5, a major hybridizing RNA species of 9.0 kb was seen with both genotypes and both samples displayed similar hybridization intensities. Probing the same blot with a mouse 8-actin cDNA also yielded hybridization signals (2.0 kb) of comparable intensities (densitome0.27 AOD; wild type: 0.35 AOD), indicattry, spa/spa:
Neuron 286
2
glycine
A
3
[log KM]
m” E .
-
2.8
I -
1.9
1
B Figure 4. Glycine-Elicited Membrane Currents Recorded from Xenopus Oocytes !njected with Spinal Cord Poly(A)’ RNA (A) Dose-response relation of glycine-induced inward currents in oocytes clamped at -70 mV. A single sigmoid fit of normalized glycine-evoked peak currents gave half-maximal responses for spastic homozygotes (closed circles) at 180 PM and for control mice (open circles) at 230 uM glycine. Inserted traces show responses for 200 PM (upper trace) and 1 mM glycine (lower trace) from oocytes injected with control and spa/spa poly(A)’ RNA, Bars reflect duration of agonist application. (B) Inhibition of glycine responses by strychnine. Peak current responses obtained at 200 PM glycine of oocytes injected with either spastic (closed circles) or control (open circles) poly(A) RNA were determined in the presence of increasing antagonist concentrations. lCs0 values of strychnine inhibition were 22 nM for control and 12 nM for spastic poly(A)’ RNA. Data are normalized to current responses obtained in the absence of inhibitor.
ing that expression levels were quantitatively similar in both homozygotic spastic and wild-type animals (data not shown). Thus, al subunit transcript levels appear to be not significantly affected by the mutation. Cell cultures from fetal mouse spinal cord express high levelsof GlyRN butalmost no GlyRA protein (Hoch et al., 1989). In primary cultures prepared from spa/spa fetuses, immunoassays employing MAb 4a revealed total glycine receptor contents similar to those of control cultures (spa/spa: 0.110 + 0.017 AOD/vg; control: 0.124 + 0.024 AODlpg; standardized to membrane protein). Likewise, the apparent molecular weight of the a subunit present in these cells did not differ from that in control cultures (Figure 6). Thus, the spastic mutation was not manifest under culture conditions, which is consistent with its selective effect on GlyRA accumulation in vivo.
Figure 5. Northern Blot Analysis of al Transcripts of Homozygous spastic and Wild-Type Animals
in Spinal Cord
Poly(A)’ RNA isolated from spinal cord of homozygous spastic and wild-type animals was electrophoresed, blotted, and hybridized with a random-primed al subunit cDNA as described in Experimental Procedures. Besides a major hybridizing band of 9.0 kb, both samples displayed considerable heterogeneity in the lower molecular weight range. This is consistently seen with glycine al subunit mRNA and probably reflects the presence of multiple transcripts (Crenningloh et al., 1987; Malosio et al., 1991a, 1991b). Positions of size markers (kb) are indicated.
Discussion Heterogeneity of neurotransmitter receptors is now recognized as a widespread phenomenon throughout the nervous system (Betz, 1990). By use of selective antibody and radioligand probes, two developmentally regulated isoforms of the inhibitory glycine receptor, GlyR* and GI~RN, are distinguished in rodent spinal cord. The major finding of this paper is that only GI~RA, and not GlyRN, levels are reduced in the mutant mouse spastic. This is evident from a strong reduction of MAb 4a immunoreactivity in adult, but not newborn, spastic animals. Moreover, low [3H]strychnine binding activity, reduced MAb 2b immunoreactivity, and normal electrophysiological prop erties of the glycine receptors induced by heterologous expression of spa/spa poly(A)’ RNA all are consistentwith our previous analysis,which indicated
Clyne
Receptor
lsoforms
in the spashc
Mouse
187
C
92.5K
-
66.2K
-
45.OK
-
31.OK
-
Figure 6. Clycine from Fetal Control
sion of other glycine receptor genes essential for biosynthesis or turnover of the GI~RA multi-subunit complex. While not required for heterologous expression of glycine receptor channels in Xenopus oocytes (Schmieden et al., 1989) and mammalian cells (Sontheimer et al., 1989), a reduction in levels of the nonligand-binding B polypeptide (Crenningloh et al., 1990; Malosio et al., 1991a) may reduce the stability of
S
-
Receptor in Spinal Cord and spa/spa Mice
Cultures
49K
Dissociated
Western blot analysisof membranes prepared from thecultured cells was performed using MAb 4a. C, control; S, spa/spa.
reduced levels, but normal functional properties, of the receptor found in the adult mutant (Becker et al., 1986). In addition, the spastic mutation was without effect under culture conditions known to favor GI~RN, but not GlyR,,, expression (Hoch et al., 1989). Our analysis of the spastic mutant corroborates the functional importance of distinct glycine receptor isoforms and shows that a selective deficiency of a developmentally regulated receptor isoform may provide a molecular basis for the age-dependent manifestation of a neurological disorder. Indeed, the postnatal appearance of spastic symptoms in spa/spa mice (Chai, 1961; Chai et al., 1982) coincides with replacement of GlyRN by GlyRA in rodent spinal cord. In homozygotic mutants, the late onset of the spastic phenotype most likely reflects the insufficient expression of GlyRA after postnatal day 10, i.e., a time when GI~RN levels are already low. This is reminiscent of other hereditary diseases that selectively affect developmentally regulated protein isoforms, e.g., altered globin gene expression resulting in thalassemia (Weatherall and Clegg, 1982). The mechanism by which the spastic gene affects accumulation of GlyR,+ but not GlyRN, remains to be defined, but it may involve transcriptional as well as posttranscriptional events: -Both our oocyte expression experiments and the Northern blot data indicate that al transcript levels are not altered by the spastic mutation. However, a mutation of al polypeptide structure not detectable by our analytical methods (these data; Becker et al., 1986) may interfere with the metabolic stability of GlyRA in the postsynaptic membrane. -The spastic mutation may interfere with the expres-
the GI~RA complex. In addition, a membrane-associated 93 kd protein thought to contribute to the postsynaptic anchoring of the glycine receptor has been identified (Schmitt et al., 1987; Becker et al., 1989). During development of rodent spinal cord, the 93 kd protein is coexpressed with ClyRA (Becker et al., 1988). - Epigenetic influences may appear only at the age of predominant GlyRA expression. For example, pathological alterations of intermediary metabolism may influence CNS expression of neurotransmitter receptors. Experimental induction of hyperglycinemia in neonatal rats by injection of glycine or serine has been shown to increase the postnatal accumulation of pHIstrychnine-binding sites (Benavides et al., 1981). However, amino acid contents in serum (C.-M. Becker, unpublished data) and brain (Chai et al., 1962) of adult spa/spa miceare not significantly different from those of control littermates. Autoantibodies have been implicated in nicotinic acetylcholine receptor downregulation in myasthenia gravis (Drachman et al., 1978), but serum from spastic homozygotes does not contain detectable amounts of immunoglobulin reactive with purified glycine receptor (C.-M. Becker, unpublished data). At present, noneof the glycine receptor subunit genes has been mapped in the mouse genome. Elucidation of the relation between the spastic locus on mouse chromosome 3 (Either and Lane, 1980) and the chromosomal representation of glycine receptor subunits should provide information about the involvement of glycine receptor genes in this murine neurological disorder. Thespasticmutationofthe mousefindsclosecorrelates in bovine and human diseases. Inherited myoclonus of Poll Hereford calves is characterized by a severe loss of [3H]strychnine-binding sites from spinal cord (Cundlach, 1990). However, motor symptoms are already manifest in fetal calves (Healy et al., 1985), suggesting a prenatal defect in glycine receptor expression. Furthermore, different disease states in man show symptoms resembling those of the spa/spa mouse, e.g., hyperekplexia (Heller and Hallett, 1982; Saenz-Lope et al., 1984) and, possibly, some types of spastic paraplegia (Harding, 1981). The analogy of the murine spastic syndrome to these human diseases certainly warrants further research. Experimental
Procedures
Animal Care Newborn spa/spa mice are phenotypically indistinguishable from the wild type (+/+) and were therefore obtained from matings of parent spa/spa mice bred on a 66C3Feala hybrid background (Jackson Laboratory, Bar Harbor, ME). The yield was low,
NWVX! 288
as it is difficult to obtain spastic litters (Chai, 1961; Chai et al., 1962; Either and Lane, 1980). Animals were removed from litters at indicated ages. Age-matched control animals were obtained by matings of heterozygous (spa/+) to wild-type (+/+) hybrid mice, or were wild type (+I+) of the C57BU6J strain. Membrane Preparation Spinal cord, including medulla oblongata and pons, was dtssected under a binocular microscope. The tissue was immediately frozen in liquid nitrogen and stored at -7OOC. Crude membrane fractions were prepared as described (Becker et al., 1989). Briefly, tissue was homogenized in 20 vol of ice-cold 50 mM Tris-HCI (pH 7.4), containing 5 mM EDTA, 5 mM ECTA, and an extended cocktail of protease inhibitors (Becker et al., 1988). After centrifugation and repeated washing, membranes were suspended in 25 mM KPi (pH 7.4), containing 200 mM KCI and protease inhibitors. The protein content of membrane fractions was assayed using a modified Lowry method (Larson et al., 1986). Immunological Methods and [‘HIStrychnine Binding Assay For determination of MAb 4a immunoreactivity, crude spinal cord membranes were subjected to Western blot analysis (10% acrylamide gel, 75 ug of protein per lane) or a dot receptor immunoassay (IO wg of protein per well) as described (Becker et al., 1989). Binding of MAb 2b, which recognizes an epitope of the native glycine receptor al subunit, was determined in a membrane immunoassay using IO kg of protein per well (Becker et al., 1988). All determinations were performed in triplicate, and specific immunoreactivities were corrected for absorbances ohtained in the absence of first antibody. lmmunoassays on membrane samples from control mice expressing adult levels of glytine receptor antigen produced signal to background ratios ranging from ~3.0 to ~6.4. G’lycine-displaceable binding ot J3H]strychnine (IO nM, 29 Ciimmol; Amersham, Braunschweig, Germany) to crude spinal cord membranes (100 ug of protein) was determined in triplicate by a small-scale filtration assay (Becker et al., 1986). Xenopus Oocyte Expression and Northern Blot Analysis PolytA)’ RNA was isolated from mouse spinal cord using the Fast Track system (Invitrogen, San Diego, CA). Injection into Xenopus oocytes and voltage-clamp recordings were done as previously described (Schmieden et al., 1989). For Northern blot analysis, poly(A)+ RNA preparations (5.5 pg per lane) were electrophoresed on a 1.4% agarose gel containing 6% (w/v) formaldehyde. After blotting to Hybond N (Amersham) and UV cross-linking, hybridization was performed with a 12P-labeled, random-primed EcoRl fragment of clone uI,,,~, (Malosio et al., 1991b) in 6x SSC, 5x Denhardt’s solution, 0.5% SDS at 62’C. Washings were in 2x SSC, 0.1% SDS at room temperature and 2x SSC, 0.1% SDS at 62OC. After autoradiography (6 days), the blot was rehybridized to a random-primed mouse B-actin cDNA under the same conditions. Autoradiographic signals were quantified by scanning in a Hirschmann Elscript 400 densitometer. Cell Culture In a few instances, termed pregnancies were obtained in spa/spa mice from matingstospa/spa males, and dissociated cell cultures were prepared on day 13.5 of gestation (Hoch et al., 1989). In the experiment shown, fetuses of spastic genotype were acquired from a single maternal spa/spa mouse. Cells were plated at a density of 5 x IO6 per dish (6 cm diameter) and cultured for 3 weeks. Parallel cultures from wild-type fetuses of 3 maternal mice served as a control. Crude membranes were prepared from the harvested cells (Hoch et al., 1989) and subjected to Western blot employing MAb 4a. Total glycine receptor contents were determined by immunoassaywith MAb4a.Datagiven inthetext are means k SEM of 4 culture dishes for each genotype.
was supported
Received
August
29, 1991; revised
November
20, l’l’i :
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Acknowledgments This work
schaft (SFB317, Leibnizand Heisenbergprograms). P. I. rrcerved an EMBO short-term fellowship. We thank J. Yazdanran and M. Hertel for animal care, I. Walters for expert technical assrstantr, W. Hoch, K. Nave, and T. Prosper0 for a critical reading of the manuscript, and I. Baro for skillful help during its preparation. The costs of publication of this article were defraved In part by the payment of page charges. This article must theretore be hereby marked “advertisement” in accordance with 18 USC Set tion 1734 solely to indicate this fact.
by the Deutsche
Forschungsgemein-
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Larson, E., Howlett, B., and Jagendorf, A. (1986). Artificial reductant enhancement of the Lowry method for protein determination. Anal. Biochem. 755, 243-248. Malosio, (1991a). mRNAs 2409. Malosio, Schmitt, generates receptor.
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B., Kuhse, J., and Betz, H. of glycine receptor subunit rat brain. EMBO J. 70,2401-
M.-L., Crenningloh, G., Kuhse, J., Schmieden, V., B., Prior, P., and Betz, H. (1991b). Alternative splicing two variants of the al subunit of the inhibitory glycine J. Biol. Chem. 266, 2048-2053.
Pfeiffer, F., Simler, R., Crenningloh, C., and Betz, H. (1984). Monoclonal antibodies of the glycine receptor of rat spinal cord. Proc. Natl. Acad. Sci. USA 87, 7224-7227. Rienitz, A., Becker, C.-M., Betz, H., and Schmitt, B. (1987). The chloride channel blocking agent, t-butyl bicyclophosphorothionate, binds to the gamma-aminobutyric acid-benzodiazepine, but not to the glycine receptor in rodents. Neurosci. Lett. 76,9195. Saenz-Lope, E., Herranz-Tanarro, Pena, J. R. (1984). Hyperekplexia: startle responses. Ann. Neurol. Schmieden, V., Grenningloh, (1989). Functional expression nine binding 48 kd subunit 695-700.
F. J., Masdeu, a syndrome 75, 36-41.
J. C., and Chacon of pathological
C., Schofield, P. R., and Betz, H. in Xenopus oocytes of the strychof the glycine receptor. EMBO J. 8,
Schmitt, B., Knaus, P., Becker, C.-M., and Betz, H. (1987). MC93000 polypeptide of the postsynaptic glycine receptor peripheral membrane protein. Biochemistry 26, 805-811.
The is a
Schroder, S., Hoch, W., Becker, C.-M., Crenningloh, C., and Betz, H. (1991). Mapping of antigenic epitopes on the al subunit of the inhibitory glycine receptor. Biochemistry 30, 42-47. Sontheimer, H., Becker, C.-M., Pritchett, D. P., Schofield, P. R., Crenningloh, C., Kettenmann, H., Betz, H., and Seeburg, P. H. (1989). Functional chloride channels by mammalian cell expression of rat glycine receptor subunit. Neuron 2, 1491-1497. Weatherall, D. J., and Clegg, Cell 29, 7-9.
J. B. (1982). Thalassemia
White, W. F. (1985). The glycine spastic (spa): strychnine binding ogy. Brain Res. 329, 1-6.
revisited.
receptor in the mutant mouse characteristics and pharmacol-
White, W. F., and Heller, A. H. (1982). Glycine receptor in the mutant mouse spastic. Nature 298, 655-657.
alteration
Vol. 8, 283-289,
February,
1992, Copyright
0 1992 by Cell Press
Isoform-Selective Deficit of Clycine in the Mouse Mutant spastic Cord-Michael Becker,*+ Volker Paola Tarroni,*§ Uta Sfrasser,* and Heinrich Betz*
Schmieden,*
*Zentrum fiir Molekulare Biologie +Neurologische UniversiGtsklinik UniversitBt Heidelberg Im Neuenheimer Feld 282 D-6900 Heidelberg *Max-Planck-lnstitut ftir Hirnforschung DeutschordenstraBe 46 D-6000 Frankfurt 71 Germany
Summary The mutant mouse spastic(spa) develops a characteristic motor disorder about 2 weeks after birth, with symptoms resembling sublethal poisoning by the glycinergic antagonist strychnine. Correspondingly, adult homozygotic mutants (spa/spa) exhibit a severe reduction of inhibitory glycine receptors in spinal cord and brain. Here we show that the spastic mutation selectively interferes with the postnatal accumulation of the adult isoform of the glytine receptor protein, whereas perinatal expression of the neonatal receptor isoform is not detectably affected. Heterologous expression in X. laevis oocytes of poly(A)+ RNA and Northern blot analysis indicate normal levels of glycine receptor al subunit transcripts in spinal cord of adult spastic mutants. Thus, the age-dependent manifestation of spastic symptoms after birth reflects a selective effect of the mutation on the developmental expression of the adult glycine receptor isoform. Introduction Mice homozygous for the recessive spastic mutation (genotype spa/spa) on chromosome 3 (Either and Lane, 1980) develop a severe motor disorder during the second postnatal week(reviewed by Becker, 1990). The syndrome is characterized by muscle rigidity, tremor, and myoclonic jerks (Chai, 1961; Chai et al., 1962) and electromyographically resem bles poisoning by subconvulsive doses of strychnine (Heller and Hallett, 1982), an antagonist of the inhibitory glycine receptor (reviewed by Betz and Becker, 1988; Langosch et al., 1990). Indeed, both glycine receptor levels determined by high affinity [3H]strychnine binding (White and Heller, 1982; White, 1985; Becker et al., 1986; Rienitz et al., 1987) and glycine-mediated chloride conductance recorded electrophysiologically(Biscoe and Duchen, 1986) are drastically reduced in spa/spa mice. However, the ligand binding properties of the glycine
5 Permanent address: Centro di Farmacologia Cellulare, Dipartimento di Farmacologia Medica, Universite di Milano, Via Vanvitelli 32, l-20129 Milano, Italy.
Receptors
receptor such as affinity for [3H]strychnine and the agonistic amino acids glycine, B-alanine, and taurine are not significantly affected by the mutation (White, 1985; Becker et al., 1986). Antigenic epitopes, subunit composition, and synaptic localization of the glycine receptor complex are likewise conserved in spinal cord of adult mutant mice (Becker et al., 1986). Glycine receptors from rodent spinal cord are composed of a and p subunits. The amino acid sequences of these polypeptides deduced from corresponding cDNAs are highly homologous and share a large, extracellular N-terminal domain followed by four hydrophobic transmembrane regions. Their predicted transmembrane structure characterizes the glycine receptor subu nits as typical members of a superfamily of genes encoding ligand-gated ion channels (reviewed by Betz, 1990). Differential expression of distinct variants of the ligand-binding a subunit (Grenningloh et al., 1987; Kuhse et al., 1990a, 199Ob, 1991; Malosio et al., 1991a) contributes to heterogeneity of the receptor protein during postnatal development (Becker et al., 1988). A neonatal isoform of the glycine receptor (GlyRN) is abundantly expressed in spinal cord tissue of newborn rats (Becker et al., 1988) and in primary cultures of fetal mouse spinal neurons (Hoch et al., 1989). CI~RN exhibits only low affinity for strychnine and contains a ligand-binding subunit (a2) of 49 kd as an integral membrane-spanning polypeptide (Becker et al., 1988; Hoch et al., 1989; Kuhse et al., 1990a, 1991). GI~RN was originally identified using monoclonal antibody (MAb) 4a, which recognizes a peptide epitope common to all glycine receptor a subunits variants known (Pfeiffer et al., 1984; Becker et al., 1988; SchrBder et al., 1991). In contrast, the adult isoform of the receptor (GI~RA) binds strychnine with high affinity. GlyRn is selectively recognized by MAb 2b, which binds to an epitope at the N-terminus of the al subunit (Schrdder et al., 1991). GI~RA is thought to be a pentamer composed of an al subunit of 48 kd and a structural p subunit of 58 kd (Langosch et al., 1990). The function of the non-ligand-binding fl polypeptide is not fully understood, as heterologous expression of glycine receptorasubunitscreatesglycine-gatedchloridechannels (Schmieden et al., 1989; Sontheimer et al., 1989; Kuhse et al., 1990a, 1991). Recent cloning experiments have revealed the existence in rodent CNS of additional a subunit variants (a3 and a4), suggesting further complexity of glycine receptors (Kuhse et al., 1990b, 1991; Y. Maulet, B. Matzenbach, and H. Betz, unpublished data). However, mRNA levels in spinal cord of these novel a subunit variants are low (Malosio et al., 1991a), indicating that they correspond to minor receptorisoforms.Thus,theantibodyand radioligand probes described above permit a reliable analysis of the major glycine receptor isoforms of rodent spinal cord, GlyRN and GlyRA (Becker et al., 1988, 1989).
NlYJr0ll 284
A E
0.80
a E
0.60
Here, we have used MAbs directed against glycine receptor a subunits to analyze the expression of glytine receptor subpopulations in the adult and developing spastic mouse mutant. Our data show that the spastic phenotype of the mutant correlates with a dramatic reduction in spinal cord levels of ClyRA, but not GlyRN.
? ‘S 0.40 l? t 0.20
Moreover, the developmental appearance of symptoms coincides with the postnatal switch from neonatal to adult glycine receptor isoforms. Our data suggest a selective deficit in the regulation of ClyRA expression, which results in the age-dependent manifestation of a neurological disease.
spastic natal (calculated)
age
[postnatal
days]
Results
0
5
10 age
C
15 [postnatal
20
adult
days]
1800,
I
1500.
age Figure
1. Postnatal
Development
[postnatal of Glycine
days] Receptor
lsoforms
(A) Total glycine receptor content of rat spinal cord (open squares) was assessed by MAb 4a immunoreactivity. ClyR,, levels (open circles) were determined by high affinity [‘HIstrychnine binding. MAb 4a immunoreactivity and pHIstrychnine binding per mg of membrane protein were normalized to values found in adult animals, in which only GlyR, is detected (Becker et al., 1988). The difference between these values yielded the fraction of low affinity antagonist-binding receptors, i.e., ClyRN (closed squares). For each age, dataarethe means + SEM of 3 membrane preparations pooled from 2 rats, each. Values for adult rats were as follows: specific MAb 4a immunoreactivity, 41.8 k 6.6 AOD per mg of protein; [3H]strychnine binding, 1.32 f 0.28 pmol per mg of protein (data from Becker et al., 1988). (B) Postnatal accumulation of MAb 4a antigen in spinal cord of control (open circles) and spa/spa mice(closed circles). Antibody binding to spinal cord membranes was determined by immunoassay. (C) Postnatal expression of ClyRn as determined by binding of MAb 2b to spinal cord membranes from control mice (open circles) and spastic homozygotes (closed circles). In (B) and (C), data represent the mean f SEM of triplicate determinations of MAb 4a and MAb 2b immunoreactivities. The
Total glycine receptor levels were determined in rodent spinal cord at different postnatal ages by immunoassay employing MAb 4a, which defines an epitope common to all glycine receptor a subunits (Schroder et al., 1991). Bycomparison with selective markers of GlyRA, e.g., MAb 2b and high affinity t3H]strychnine binding (Becker et al., 1988), relative levels of GlyR,, and GI~RN were calculated from these data (Figure IA). In rat spinal cord, the postnatal accumulation of [3H]strychnine-binding sites and of MAb 2b antigen (data not shown; see Becker et al., 1988) proceeded in parallel, thus confirming their association with GlyRA (Becker et al., 1988). At birth, GlyRN accounted for about 70% of the total glycine receptor protein present. Thereafter, GI~RA increased at the expense of GI~RN, resulting in acomplete exchange of the neonatal isoform after about 2 weeks of age (Figure IA). Glycine receptor preparations purified from adult spa/spa mice display antigenic and ligand binding properties indistinguishable from those of the receptor isolated from phenotypically normal littermates (Becker et al., 1986). Thus, adult homozygous mutants express GI~RA, though at drastically reduced levels. This prompted us to study the effect of the spastic mutation on GI~RN expression. In spinal cord of control mice, the content of MAb 4a-reactive total glycine receptor increased about 60% within 1 week after birth and, thereafter, slowly declined to levels seen at birth (Figure IB). During the second postnatal week, MAb 2b immunoreactivity sharply increased from close to background values to mature levels (Figure IC). This indicates that, in the mouse, GlyRN is replaced by GlyR* at a time corresponding to that reported above for rat spinal cord development. In spastic homozygotes, the postnatal accumulation of MAb 4a and MAb 2b immunoreactivities was dramatically altered. Although spa/spa animals possessed normal amounts of MAb 4a antigen at birth, a severe reduc-
same membrane samples were applied to both immunoassays, each containing the pooled tissue from 2 mice of the indicated age and genotype. This experiment was repeated on independently collected tissue samples with essentially the same results.
Glycine
285
Receptor
lsoforms
in the spast~
Mouse
S
C h ,Z > 'Z $ L
ad
1.0
neo
neo
ad
0.8 0.6
: .f;r
0.4
T
0.2
J
4gK
-\
48K
00
neonatal Figure 2. Clycine Receptor nine Binding in Spinal Cord and spastic Homozygotes
adult lmmunoreactivity and [-‘H]strychof Neonatal and Adult Control Mice
Specific binding of MAbs 4a and 2b and of [3H]Strychnine was normalized to values obtained with adult control mice (immunoreactivities of 68.9 * 7.6 AOD per mg of protein for MAb 4a and 22.5 f 3.8 AOD per mg of protein for MAb 2b; [‘HIstrychnine binding of 934 f 111 fmol per mg of protein). Data are means f SEM of separate membrane samples (n = 2 for neonates; n = 3 for adult mice), each containing tissue from 2 mice of the indicated age and genotype. Open bars, neonatal (postnatal day 0) and adult control mice; closed bars, spastic homozygotes. Animals are different from those employed for Figure 1. Asterisks indicate that the adult spastic micediffered from control animals at p < 0.01 by the two-tailed Student’s t test for unpaired samples.
tion occurred thereafter, resulting in low adult levels (Figure IB). MAb 2b immunoreactivity indicative of GI~RA was drastically reduced in spa/spa animals throughout postnatal life (Figure ICI). Apparently, the spastic mutation has no major effect on prenatal receptor protein accumulation, suggesting that GI~RN expression proceeds normally. Using a larger sample of spinal cords from newborn spa/spa mice, the MAb 4a antigen could be identified as GI~RN by the following criteria: first, crude memneonates did not react with MAb branes from spa/spa 2b (Figure 2; compare also Figure IC); second, high affinity [3H]strychnine binding to these membranes was low, consistent with a prevalence of GI~RN (Figure 2); finally, Western blot analysis employing MAb 4a revealed a49 kd protein (Figure 3). This corresponds to the apparent molecular weight of the ligand-binding subunit of GlyRN (Becker et al., 1988; Hoch et al., 1989). In contrast to the situation at birth, membranes mice showed a significant reducfrom adult spa/spa tion in MAb4a antigen compared with control animals (Figure 2). Western blot analysis (Figure 3) revealed a polypeptide of 48 kd, i.e., the previously described al subunit of GI~RA (Becker et al., 1988; Pfeiffer et al., 1984). This is consistent with earlier observations on purified glycine receptor preparations isolated from mice (Becker et al., 1986). In accordance adult spa/spa with the low levels of MAb 2b immunoreactivity in adult mutants, [3H]strychnine binding showed only homozygotes during postnalittle increase in spastic tal life (Figure 2). These data suggest that the spastic mutation selectively affects accumulation of GlyR,+ but not GlyRN.
Figure 3. Western Blot Analysis of Clycine natal and Adult Control and spa/spa Mice
Receptor
from
Neo-
Membrane preparations were subjected to SDS-polyacrylamide gel electrophoresis and reacted with MAb 4a. Stained bands of -46 kd (adult), ~32 kd, and ~34 kd (neonatal) most likely resulted from limited proteolysis despite the use of an extended cocktail of protease inhibitors (see also Becker et al., 1988). C, control; S, spa/spa mice. Neonatal (neo), postnatal day 0; adult (ad), &weekold mice.
Xenopus laevis oocytes injected with poly(A)+ RNA isolated from rodent spinal cord express functional glycine receptors (Schmieden et al., 1989). After injection of spinal cord poly(A)’ RNA from either adult homozygotes, glycinecontrol mice or adult spastic induced inward currents were revealed by voltageclamp recording in the majority of the injected oocytes, with half-maximal responses at 180 uM and 230 uM glycine, respectively (Figure 4A). For both doseresponse relations, Hill coefficients of 3.0 were derived. Glycine currents reversed at about -25 mV, i.e., the equilibrium potential of chloride (data not shown). Variations in maximal current between different Xenopus oocytes observed with the same batch of poly(A)‘RNA, however, precluded a precisequantitation of glycine receptor transcript levels. The glycine responses obtained with both control and spa/spa poly(A)‘RNA were sensitive to strychnine, with a halfmaximal inhibition at 22 nM and 12 nM, respectively (Figure 4B). These data confirm the notion that the propertiesof GlyR~are notaltered bythespastic mutation (Becker et al., 1986). mutation alters To investigate whether the spastic the levels of glycine receptor al subunit transcripts in adult animals, we performed a Northern analysis of spinal cord poly(A)+ RNA isolated from homozygotic spastic and wild-type animals. As shown in Figure 5, a major hybridizing RNA species of 9.0 kb was seen with both genotypes and both samples displayed similar hybridization intensities. Probing the same blot with a mouse 8-actin cDNA also yielded hybridization signals (2.0 kb) of comparable intensities (densitome0.27 AOD; wild type: 0.35 AOD), indicattry, spa/spa:
Neuron 286
2
glycine
A
3
[log KM]
m” E .
-
2.8
I -
1.9
1
B Figure 4. Glycine-Elicited Membrane Currents Recorded from Xenopus Oocytes !njected with Spinal Cord Poly(A)’ RNA (A) Dose-response relation of glycine-induced inward currents in oocytes clamped at -70 mV. A single sigmoid fit of normalized glycine-evoked peak currents gave half-maximal responses for spastic homozygotes (closed circles) at 180 PM and for control mice (open circles) at 230 uM glycine. Inserted traces show responses for 200 PM (upper trace) and 1 mM glycine (lower trace) from oocytes injected with control and spa/spa poly(A)’ RNA, Bars reflect duration of agonist application. (B) Inhibition of glycine responses by strychnine. Peak current responses obtained at 200 PM glycine of oocytes injected with either spastic (closed circles) or control (open circles) poly(A) RNA were determined in the presence of increasing antagonist concentrations. lCs0 values of strychnine inhibition were 22 nM for control and 12 nM for spastic poly(A)’ RNA. Data are normalized to current responses obtained in the absence of inhibitor.
ing that expression levels were quantitatively similar in both homozygotic spastic and wild-type animals (data not shown). Thus, al subunit transcript levels appear to be not significantly affected by the mutation. Cell cultures from fetal mouse spinal cord express high levelsof GlyRN butalmost no GlyRA protein (Hoch et al., 1989). In primary cultures prepared from spa/spa fetuses, immunoassays employing MAb 4a revealed total glycine receptor contents similar to those of control cultures (spa/spa: 0.110 + 0.017 AOD/vg; control: 0.124 + 0.024 AODlpg; standardized to membrane protein). Likewise, the apparent molecular weight of the a subunit present in these cells did not differ from that in control cultures (Figure 6). Thus, the spastic mutation was not manifest under culture conditions, which is consistent with its selective effect on GlyRA accumulation in vivo.
Figure 5. Northern Blot Analysis of al Transcripts of Homozygous spastic and Wild-Type Animals
in Spinal Cord
Poly(A)’ RNA isolated from spinal cord of homozygous spastic and wild-type animals was electrophoresed, blotted, and hybridized with a random-primed al subunit cDNA as described in Experimental Procedures. Besides a major hybridizing band of 9.0 kb, both samples displayed considerable heterogeneity in the lower molecular weight range. This is consistently seen with glycine al subunit mRNA and probably reflects the presence of multiple transcripts (Crenningloh et al., 1987; Malosio et al., 1991a, 1991b). Positions of size markers (kb) are indicated.
Discussion Heterogeneity of neurotransmitter receptors is now recognized as a widespread phenomenon throughout the nervous system (Betz, 1990). By use of selective antibody and radioligand probes, two developmentally regulated isoforms of the inhibitory glycine receptor, GlyR* and GI~RN, are distinguished in rodent spinal cord. The major finding of this paper is that only GI~RA, and not GlyRN, levels are reduced in the mutant mouse spastic. This is evident from a strong reduction of MAb 4a immunoreactivity in adult, but not newborn, spastic animals. Moreover, low [3H]strychnine binding activity, reduced MAb 2b immunoreactivity, and normal electrophysiological prop erties of the glycine receptors induced by heterologous expression of spa/spa poly(A)’ RNA all are consistentwith our previous analysis,which indicated
Clyne
Receptor
lsoforms
in the spashc
Mouse
187
C
92.5K
-
66.2K
-
45.OK
-
31.OK
-
Figure 6. Clycine from Fetal Control
sion of other glycine receptor genes essential for biosynthesis or turnover of the GI~RA multi-subunit complex. While not required for heterologous expression of glycine receptor channels in Xenopus oocytes (Schmieden et al., 1989) and mammalian cells (Sontheimer et al., 1989), a reduction in levels of the nonligand-binding B polypeptide (Crenningloh et al., 1990; Malosio et al., 1991a) may reduce the stability of
S
-
Receptor in Spinal Cord and spa/spa Mice
Cultures
49K
Dissociated
Western blot analysisof membranes prepared from thecultured cells was performed using MAb 4a. C, control; S, spa/spa.
reduced levels, but normal functional properties, of the receptor found in the adult mutant (Becker et al., 1986). In addition, the spastic mutation was without effect under culture conditions known to favor GI~RN, but not GlyR,,, expression (Hoch et al., 1989). Our analysis of the spastic mutant corroborates the functional importance of distinct glycine receptor isoforms and shows that a selective deficiency of a developmentally regulated receptor isoform may provide a molecular basis for the age-dependent manifestation of a neurological disorder. Indeed, the postnatal appearance of spastic symptoms in spa/spa mice (Chai, 1961; Chai et al., 1982) coincides with replacement of GlyRN by GlyRA in rodent spinal cord. In homozygotic mutants, the late onset of the spastic phenotype most likely reflects the insufficient expression of GlyRA after postnatal day 10, i.e., a time when GI~RN levels are already low. This is reminiscent of other hereditary diseases that selectively affect developmentally regulated protein isoforms, e.g., altered globin gene expression resulting in thalassemia (Weatherall and Clegg, 1982). The mechanism by which the spastic gene affects accumulation of GlyR,+ but not GlyRN, remains to be defined, but it may involve transcriptional as well as posttranscriptional events: -Both our oocyte expression experiments and the Northern blot data indicate that al transcript levels are not altered by the spastic mutation. However, a mutation of al polypeptide structure not detectable by our analytical methods (these data; Becker et al., 1986) may interfere with the metabolic stability of GlyRA in the postsynaptic membrane. -The spastic mutation may interfere with the expres-
the GI~RA complex. In addition, a membrane-associated 93 kd protein thought to contribute to the postsynaptic anchoring of the glycine receptor has been identified (Schmitt et al., 1987; Becker et al., 1989). During development of rodent spinal cord, the 93 kd protein is coexpressed with ClyRA (Becker et al., 1988). - Epigenetic influences may appear only at the age of predominant GlyRA expression. For example, pathological alterations of intermediary metabolism may influence CNS expression of neurotransmitter receptors. Experimental induction of hyperglycinemia in neonatal rats by injection of glycine or serine has been shown to increase the postnatal accumulation of pHIstrychnine-binding sites (Benavides et al., 1981). However, amino acid contents in serum (C.-M. Becker, unpublished data) and brain (Chai et al., 1962) of adult spa/spa miceare not significantly different from those of control littermates. Autoantibodies have been implicated in nicotinic acetylcholine receptor downregulation in myasthenia gravis (Drachman et al., 1978), but serum from spastic homozygotes does not contain detectable amounts of immunoglobulin reactive with purified glycine receptor (C.-M. Becker, unpublished data). At present, noneof the glycine receptor subunit genes has been mapped in the mouse genome. Elucidation of the relation between the spastic locus on mouse chromosome 3 (Either and Lane, 1980) and the chromosomal representation of glycine receptor subunits should provide information about the involvement of glycine receptor genes in this murine neurological disorder. Thespasticmutationofthe mousefindsclosecorrelates in bovine and human diseases. Inherited myoclonus of Poll Hereford calves is characterized by a severe loss of [3H]strychnine-binding sites from spinal cord (Cundlach, 1990). However, motor symptoms are already manifest in fetal calves (Healy et al., 1985), suggesting a prenatal defect in glycine receptor expression. Furthermore, different disease states in man show symptoms resembling those of the spa/spa mouse, e.g., hyperekplexia (Heller and Hallett, 1982; Saenz-Lope et al., 1984) and, possibly, some types of spastic paraplegia (Harding, 1981). The analogy of the murine spastic syndrome to these human diseases certainly warrants further research. Experimental
Procedures
Animal Care Newborn spa/spa mice are phenotypically indistinguishable from the wild type (+/+) and were therefore obtained from matings of parent spa/spa mice bred on a 66C3Feala hybrid background (Jackson Laboratory, Bar Harbor, ME). The yield was low,
NWVX! 288
as it is difficult to obtain spastic litters (Chai, 1961; Chai et al., 1962; Either and Lane, 1980). Animals were removed from litters at indicated ages. Age-matched control animals were obtained by matings of heterozygous (spa/+) to wild-type (+/+) hybrid mice, or were wild type (+I+) of the C57BU6J strain. Membrane Preparation Spinal cord, including medulla oblongata and pons, was dtssected under a binocular microscope. The tissue was immediately frozen in liquid nitrogen and stored at -7OOC. Crude membrane fractions were prepared as described (Becker et al., 1989). Briefly, tissue was homogenized in 20 vol of ice-cold 50 mM Tris-HCI (pH 7.4), containing 5 mM EDTA, 5 mM ECTA, and an extended cocktail of protease inhibitors (Becker et al., 1988). After centrifugation and repeated washing, membranes were suspended in 25 mM KPi (pH 7.4), containing 200 mM KCI and protease inhibitors. The protein content of membrane fractions was assayed using a modified Lowry method (Larson et al., 1986). Immunological Methods and [‘HIStrychnine Binding Assay For determination of MAb 4a immunoreactivity, crude spinal cord membranes were subjected to Western blot analysis (10% acrylamide gel, 75 ug of protein per lane) or a dot receptor immunoassay (IO wg of protein per well) as described (Becker et al., 1989). Binding of MAb 2b, which recognizes an epitope of the native glycine receptor al subunit, was determined in a membrane immunoassay using IO kg of protein per well (Becker et al., 1988). All determinations were performed in triplicate, and specific immunoreactivities were corrected for absorbances ohtained in the absence of first antibody. lmmunoassays on membrane samples from control mice expressing adult levels of glytine receptor antigen produced signal to background ratios ranging from ~3.0 to ~6.4. G’lycine-displaceable binding ot J3H]strychnine (IO nM, 29 Ciimmol; Amersham, Braunschweig, Germany) to crude spinal cord membranes (100 ug of protein) was determined in triplicate by a small-scale filtration assay (Becker et al., 1986). Xenopus Oocyte Expression and Northern Blot Analysis PolytA)’ RNA was isolated from mouse spinal cord using the Fast Track system (Invitrogen, San Diego, CA). Injection into Xenopus oocytes and voltage-clamp recordings were done as previously described (Schmieden et al., 1989). For Northern blot analysis, poly(A)+ RNA preparations (5.5 pg per lane) were electrophoresed on a 1.4% agarose gel containing 6% (w/v) formaldehyde. After blotting to Hybond N (Amersham) and UV cross-linking, hybridization was performed with a 12P-labeled, random-primed EcoRl fragment of clone uI,,,~, (Malosio et al., 1991b) in 6x SSC, 5x Denhardt’s solution, 0.5% SDS at 62’C. Washings were in 2x SSC, 0.1% SDS at room temperature and 2x SSC, 0.1% SDS at 62OC. After autoradiography (6 days), the blot was rehybridized to a random-primed mouse B-actin cDNA under the same conditions. Autoradiographic signals were quantified by scanning in a Hirschmann Elscript 400 densitometer. Cell Culture In a few instances, termed pregnancies were obtained in spa/spa mice from matingstospa/spa males, and dissociated cell cultures were prepared on day 13.5 of gestation (Hoch et al., 1989). In the experiment shown, fetuses of spastic genotype were acquired from a single maternal spa/spa mouse. Cells were plated at a density of 5 x IO6 per dish (6 cm diameter) and cultured for 3 weeks. Parallel cultures from wild-type fetuses of 3 maternal mice served as a control. Crude membranes were prepared from the harvested cells (Hoch et al., 1989) and subjected to Western blot employing MAb 4a. Total glycine receptor contents were determined by immunoassaywith MAb4a.Datagiven inthetext are means k SEM of 4 culture dishes for each genotype.
was supported
Received
August
29, 1991; revised
November
20, l’l’i :
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tii)+‘:.‘( cptor
Becker, C.-M., Hermans-Borgmeyer, I., Schmitt, H., .rnd Bet/. ii 11986).Theglycinereceptordeficiencyofthemutant mousespas tic: evidence for normal glycine receptor structure and localrzation. I. Neurosci. 6, 1358-1364. Becker, C.-M., Hoch, W., and Betz, H. (1988). GIycirre receptor heterogeneity in rat spinal cord during postnatal development. EMBO J. 7, 3717-3726. Becker, C.-M., Hoch, W., and Betr, H. (1989). Sensrtrvr ~mmunoassay shows selective association of peripheral and Integral tmembrane proteins of the inhibitory glycine receptor cnmplex. I. Neurochem. 53, 124-131. Benavides, I., Loper-Lahoya, (1981). Postnatal development normal and hyperglycinergic Betz, H. (1990). Lrgand-gated acid receptor superfamily.
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thramrno
glvc me ret cap channel pro
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Acknowledgments This work
schaft (SFB317, Leibnizand Heisenbergprograms). P. I. rrcerved an EMBO short-term fellowship. We thank J. Yazdanran and M. Hertel for animal care, I. Walters for expert technical assrstantr, W. Hoch, K. Nave, and T. Prosper0 for a critical reading of the manuscript, and I. Baro for skillful help during its preparation. The costs of publication of this article were defraved In part by the payment of page charges. This article must theretore be hereby marked “advertisement” in accordance with 18 USC Set tion 1734 solely to indicate this fact.
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