Contrasting Localizations of MALS/LIN-7 PDZ Proteins in Brain and Molecular Compensation in Knockout Mice*

Hidemi MisawaDagger §, Yoshimi KawasakiDagger , Jack Mellor, Neal SweeneyDagger , Kiwon JoDagger , Roger A. NicollDagger ||, and David S. BredtDagger **

From the Departments of Dagger  Physiology and  Cellular and Molecular Pharmacology, University of California at San Francisco School of Medicine, San Francisco, California 94143-0444

Received for publication, October 12, 2000, and in revised form, November 29, 2000


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Proteins containing PDZ (postsynaptic density-95, discs large, zonula occludens) domains play a general role in recruiting receptors and enzymes to specific synaptic sites. In Caenorhabditis elegans, a complex of three PDZ proteins, LIN-2/7/10, mediates basolateral targeting of a receptor tyrosine kinase. Homologs of these LIN proteins have also been identified in higher organisms, and here we analyze the MALS/Veli (mammalian LIN-7/vertebrate homolog of LIN-7) proteins in brain. Immunohistochemical staining and in situ hybridization show that MALS occur differentially in discrete populations of neurons throughout the brain. Most neurons express only one MALS protein, although some cells contain two or even all three MALS isoforms. At the subcellular level, MALS proteins are found in both dendritic and axonal locations, suggesting that they may regulate processes at both pre- and postsynaptic sites. Targeted disruption of MALS-1 and MALS-2 does not yield a detectable phenotype, and hippocampal synaptic function and plasticity are intact in the MALS-1/2 double knockouts. Interestingly, MALS-3 protein is dramatically induced in the MALS-1/2 double knockouts, implying that dynamic changes in protein expression may play an important regulatory role for this family of synaptic PDZ proteins.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Neuronal development and function require polarized sorting of protein complexes to appropriate cellular domains within neurons. At synapses neurotransmitter receptors must align at the postsynaptic density (PSD)1 opposed to the presynaptic active zone. Although mechanisms for protein trafficking to synapses and other specializations in neurons remain poorly understood, recent work identifies a general role for proteins containing PDZ (postsynaptic density-95, discs large, zonula occludens) domains in synaptic targeting (1-3).

PDZ motifs are modular 80-amino acid domains that mediate protein-protein interactions in a variety of cellular contexts. PDZ domains contain a conserved peptide-binding grove that associates with the extreme C terminus of interacting protein ligands or with appropriate internal binding motifs (4, 5). In addition to binding to certain receptors, PDZ-containing complexes also recruit cytosolic signaling enzymes to appropriate plasma membrane domains (1-3).

Genetic studies of invertebrates have shown that PDZ proteins often play essential roles in regulating cellular signaling pathways. Mutations of Drosophila discs large, a membrane-associated guanylate kinase related to PSD-95, cause overgrowth of imaginal discs and abnormalities of larval neuromuscular junction (6, 7). In Caenorhabditis elegans, mutations of a set of three PDZ proteins, LIN-2, LIN-7, and LIN-10, disrupt differentiation of vulval precursor cells and yield a vulvaless phenotype (8). A series of elegant biochemical and cell biological studies showed that LIN-2/7/10 form a stable complex in which LIN-2 directly binds to both LIN-7 and LIN-10 (8). These interactions do not involve the PDZ domains, which remain free to interact with other cellular proteins (9). Specifically, the PDZ domain from LIN-7 binds the extreme C terminus of LET-23 and mediates basolateral localization of this receptor.

The LIN-2/7/10 complex also occurs prominently in neurons. Disruption of lin-10 in C. elegans prevents proper postsynaptic sorting of the GLR-1 glutamate receptor (10). In mammals, close homologs of LIN-2 (CASK), LIN-7 (MALS/Veli), and LIN-10 (Mint/X11) have all been identified, and the LIN-2/7/10 complex occurs at highest levels in brain (9, 11, 12). However, specific roles for the LIN-2/7/10 complex in brain remain uncertain. Some studies have suggested that this complex regulates presynaptic functions (9), whereas others have implicated postsynaptic roles (12). Immunohistochemical studies show CASK is ubiquitous in brain and occurs at both pre- and postsynaptic sites (13, 14). Mint has also been found in numerous neuronal populations in brain. The cellular localizations for MALS in brain remain unknown.

Here, we have developed antisera to the family of MALS/Veli (mammalian LIN-7/vertebrate homolog of LIN-7) homologs. Immunohistochemical staining of brain shows a heterogeneous distribution for these proteins, such that each MALS shows discrete localization in specific neuronal populations and both dendritic and axonal sites are labeled. In general, cells are endowed with only one MALS protein; however, certain neurons express multiple MALS isoforms. Targeted disruption of MALS-1 and MALS-2 does not yield a detectable phenotype, and synaptic function in hippocampus is apparently intact in the double knockouts. Disruption of MALS-1/2 proteins results in selective up-regulation of MALS-3. This differential localization and dynamic induction of MALS suggest that protein expression is an important point for regulation of this family of synaptic PDZ proteins.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation of MALS-1 Genomic DNA and Construction of Targeting Vector-- To isolate mouse MALS-1 cDNA, the N-terminal 250 bp was PCR-amplified from first-strand mouse brain cDNA using primers based on the human MALS-1 sequence (12). This mouse cDNA probe was used to isolate a bacterial artificial chromosome clone from a 129Sv/J mouse genomic library (Genome Systems, St. Louis, MO). The position of a 119-bp protein coding exon (second exon; +83-201 bp from the initiation ATG codon) was determined by DNA sequencing. The targeting vector was constructed using the pPNT replacement vector. A 1-kb intronic region downstream from the targeted exon was PCR-amplified, digested with XhoI and NotI, and subcloned into the XhoI/NotI sites of pPNT. Similarly, a 6-kb genomic region upstream to the exon was PCR-amplified, digested with KpnI and NheI, and inserted into the KpnI/XbaI sites of the pPNT vector. PCR primers used were TTACTCGAGGATCCGGACGCCGAGAATGTAACAACTG and TAAGCGGCCGCCCAGCCTTTGATTAACTTACAGGTTG for the 1-kb region and GAGTTAGGTACCCAACTCTTCCTTATCAGCTCTTTTCC and CACTTAGCTAGCGAACACAACCTCCCTAATTAATCTC for the 6-kb region. In the targeting vector (MALS-1 LH6SH1), the second exon and 2.2 kb of flanking sequence were replaced with a neo-cassette. The HSV-tk gene was included to allow for negative selection of nonrecombinants.

Generation of MALS-1 Null Mice-- The targeting vector was linearized with NotI and ligated to a NotI-compatible cap oligonucleotide to seal the ends of the linear fragment (15). This capped targeting construct was electroporated into JM-1 ES cells, and clones resistant to G418 and gancyclovir were analyzed by PCR using primers located in the neo-cassette and outside of the 1-kb intronic region: GCTAAAGCGCATGCTCCAGACTG and GGTGACTTGCCATAAAACTAGC. PCR-positive clones were further analyzed by Southern blotting both with probes containing genomic sequences outside of the targeting vector and with a neo probe. Three properly targeted clones were obtained from 282 analyzed. Targeted cells were injected into blastocysts from C57BL/6J mice and transferred into pseudopregnant CD1 female mice. Male chimeras were mated with Black Swiss (Taconic, Germantown, NY) or C57BL/6J (Jackson Laboratory, Bar Harbor, ME) females for transmission of the mutated allele through the germ line. Heterozygous mice were intercrossed to generate MALS-1 null mice. The genotypes from these and subsequent matings were determined by Southern blotting or PCR using allele specific primers as follows: GTCTGGAATACTTAGTGCACTTAG and CTGTACAAAACTCACTCTGAAGC for normal allele; CTATGACTGGGCACAACAGAC and CGTCAAGAAGGCGATAGAAGG for targeted allele. The null phenotype was confirmed by Western blotting of brain homogenates with antibodies to MALS-1.

Isolation of MALS-2 Gene and Generation of MALS-2 Null Mice-- Isolation and characterization of the MALS-2 bacterial artificial chromosome clone and construction of the MALS-2 targeting vector followed the protocol for MALS-1. The six exons encoding MALS-2 occur within a 2.3-kb genomic region. This 2.3-kb region was replaced by the neomycin cassette of pPNT vector. A 1-kb genomic region upstream (EcoRI/BamHI) and a 6-kb genomic region downstream (XhoI/NotI) were subcloned into the EcoRI/BamHI and XhoI/NotI sites of pPNT, respectively. PCR primers used are GAGTTAGAATTCAAACAGGCAGCACTCCATGTTATTGG and GAGTTAGGATCCGGTACCGTGGTGCATGAGGCCCTGCCCACAC for the 1-kb region and GAGTTACTCTCTACACCCTCACAGGTCC and GAGTTAGCGGCCGCAGCGCCAAGCCAGAACCACACC for the 6-kb region. The NotI-linearized and cap-ligated targeting vector (MALS-2 SH1LH6) was electroporated into JM-1 ES cells. Two homologously recombined clones were obtained from 157 analyzed by PCR screening. PCR primers used are CAAGCAGTCCACACAGTCACC and GTTCCACATACACTTCATTCTCAG. Male chimeras were mated with C57BL/6J females. Heterozygous mice were intercrossed to generate MALS-2 null mice. PCR primers used for genotyping are: ATGGCTGCACTGGTGGAGCCG and ACCTCGCGGATGGCGGAGCAG for normal allele and GCTAAAGCGCATGCTCCAGACTG and CGGGCTAGTCTCTGGGACAC for targeted allele.

Generation of MALS-1/2 Double Null Mice-- Female MALS-1-/- mice (F3 generation on Black Swiss background) and male MALS-2 chimeric mice were mated, and MALS-1/2 double heterozygous mice were selected by Southern blotting. Intercrossing the double heterozygous mice made MALS-1/2 double null mice. For genotyping the MALS-1 targeted locus, new PCR primers were used: GCTAAAGCGCATGCTCCAGACTG and AAGATGTGGTTCCAAGCTTAAC. The other PCR primers used for genotyping are the same as those used for MALS-1 or -2 single null mice.

Antibodies and Immunoblotting-- Polyclonal antisera to all of the MALS proteins were raised by injecting rabbits with peptides coupled to keyhole, limpet, hemoryanin. The sequences of the peptides used are as follows: MALS-1, CQQTQQNHMS; MALS-2, CQHHSYTSLERSG; MALS-3, CSRFEKMRSAKRRQQT. The pan-MALS antibody, which recognizes all three MALS isoforms, was generated against a fusion protein of glutathione S-transferase and MALS-2 as described (12). For immunoblotting, tissue homogenates were resolved by SDS-polyacrylamide gel electrophoresis (70 µg/lane) and transferred to polyvinylidene difluoride membranes. Primary antibodies were diluted (0.5 µg/ml) in blocking solution containing 2% bovine serum albumin, 0.1% Tween 20 in TBS and incubated with membranes overnight at 4 °C. Immunolabeled bands were visualized using enhanced chemilluminescence.

Immunohistochemistry-- Adult mice were anesthetized with pentobarbital and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer. The brain was removed and immersed in the same fixative for 2 h at 4 °C and then cryoprotected in 20% sucrose in phosphate-buffered saline overnight at 4 °C. Free-floating sections (40 µm) were cut on a sliding microtome. Sections were blocked for 3 h in phosphate-buffered saline containing 3% normal goat serum and then incubated in the same buffer containing diluted antiserum (0.1-0.3 µg/ml) for 2 days at 4 °C. Immunohistochemical staining was performed with an avidin/biotin/peroxidase system (ABC Elite; Vector Laboratories, Burlingame, CA) and 3, 3'-diaminobenzidine (Sigma).

In Situ Hybridization-- In situ hybridization used 35S-labeled RNA probes as described (16). Because of high homology in the coding regions of MALS-1/2/3, isoform specific cRNA probes were made from 3' noncoding regions. Amplification of a 3' fragment of MALS-1 from mouse cDNA used PCR primers based on human MALS-1 (12): CACATGTCATAGGCGCTCGAAG + CTTCTTACACTGTTTCAGTTTG. This fragment was subcloned in pCR2.1 (Invitrogen, Carlsbad, CA). The resulting plasmid was digested with BamHI and XhoI, and the insert (314 bp) subcloned into pBluscript. Specific 3' regions of MALS-2 and MALS-3 cDNA were amplified with PCR primers containing BamHI or XhoI, and subcloned into pBluescript vector. The MALS-2 primers used were: ATCATCGGATCCACCACAGCTACACGTCCT and ATCATCCTCGAGAGGGTTAAAGATCTTTAAATAAGG. The MALS-3 primers used were ATCATCGGATCCGACCTAATCATTTAAAAACTTTGTG and ATCATCCTCGAGCAGAACTGTCCTCACCAACTC.

Electrophysiology-- Adult mice were anesthetized with halothane and decapitated before removing the brain and dissecting out the hippocampus. Transverse slices (300 µm) were cut using a vibratome (Leica VT1000S) at 4 °C in a high sucrose solution containing 87 mM NaCl, 25 mM NaHCO3, 75 mM sucrose, 25 mM glucose, 2.5 mM KCl, 1.25 mM NaHPO4, 0.5 mM CaCl2, 7 mM MgCl2 (equilibrated with 95% O2, 5% CO2). Slices were then incubated at 35 °C for 30 min, cooled to room temperature for 30 min, and then transferred to artificial cerebral spinal fluid containing 119 mM NaCl, 26 mM NaHCO3, 10 mM glucose, 2.5 mM KCl, 1.3 mM MgSO4, 1 mM NaHPO4, 2.5 mM CaCl2 (equilibrated with 95% O2, 5% CO2) and held for 30 min before commencing experiments. Before transfer to the recording chamber mounted on an upright Olympus BX50 WI microscope, a cut was made between CA3 and CA1 in each slice. For field experiments a bipolar tungsten stimulating electrode was placed on the surface of CA1 stratum radiatum toward the CA3 end, and a monopolar glass recording electrode containing NaCl (1 M) was inserted into CA1 stratum radiatum at a distance of ~500 µm from the stimulating electrode. Stimulus duration was 0.1 ms, and stimuli were delivered at a frequency of 0.1 Hz. Fiber volley amplitude was measured at the peak, and EPSPs were measured by making a best fit line through the initial section (20-60% of the peak amplitude) of the rising phase. For fiber volley/EPSP comparison, data were separated into four 0.1 mV fiber volley size bins. For whole cell experiments the stimulating electrode was placed in the same fashion, and recordings were made from visually identified CA1 pyramidal cells using patch electrodes with tip resistances of 2-6 MOmega filled with a solution containing 117.5 mM cesium gluconate, 5 mM CsCl, 10 mM tetraethyl ammonium-Cl, 5 mM QX314-Cl, 8 mM NaCl, 10 mM HEPES, 10 mM acetoxymethyl ester of 5,5'-dimethyl-bis-(o-aminophenoxy)ehtane-N,N,N',N',-tetraacetic acid, 4 mM MgATP, 0.3 mM NaGTP adjusted to 280 mOsm, pH 7.4. Stimulation was performed as above, and access and input resistances were monitored continuously by applying a test voltage step of -4 mV immediately before each stimulation. Magnitudes of AMPA EPSCs were measured from average traces of 30 EPSCs in each cell. The average of 30 EPSCs in the presence of D-amino-5-phosphoenovalerate (100 µM) was subtracted from the AMPA EPSC to give the NMDA EPSC in each cell. Data were filtered at 5 KHz and stored directly to disc using the Igor Pro software (Wavemetrics) and analyzed on line. Responses were filtered at 5 kHz and digitized at 10 kHz. All measurements are given as the means ± S.E. Statistical significance was tested using Student's t test.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Characterization of Antisera to MALS-1,2,3-- To map the localization of MALS proteins throughout the brain, we developed a specific antiserum to each protein. Because MALS-1,2,3 share >80% sequence identity, antigens for peptide antibodies were carefully designed to avoid cross-reaction. We first characterized the antisera by Western blotting. Homogenates from mouse brain or COS cells transfected with each of the MALS cDNAs were separated by SDS-polyacrylamide gel electrophoresis, and following transfer, blots were probed with affinity-purified antibodies to each of the MALS proteins. Previous studies have shown that MALS-1 migrates at 29 kDa and that MALS-2,3 migrate at 26 kDa (9, 12). As shown in Fig. 1, each of the MALS antibodies recognizes a band of the expected mobility in brain homogenates. We assessed specificity of the antisera by Western blotting of COS cells transfected with each MALS cDNA. MALS-1,2 are not present in untransfected COS cells but are detected in lanes from COS cells transfected with MALS-1 and MALS-2, respectively. On the other hand, MALS-3 occurs at low levels in COS cells, although the band is more intense in MALS-3 transfected cells. Thus, the MALS antisera are all isoform-specific.


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Fig. 1.   Characterization of isoform-specific antibodies for MALS proteins. Mouse brain homogenate (lane 1) or homogenates of COS cells transfected with expression constructs for MALS-1 (lane 2), MALS-2 (lane 3), or MALS-3 (lane 4) were immunoblotted with isoform-specific MALS antibodies. Lane 5 is a homogenate from COS cells transfected with an empty vector, pcDNAIII. Note that endogenous MALS-3 protein is detected in COS cells.

Differential Localization of MALS-1,2,3 in Brain-- Immunohistochemical staining and in situ hybridization of sagittal brain sections shows a heterogeneous cellular distribution for each MALS protein and mRNA (Fig. 2). MALS-1 protein occurs at highest levels in cerebellum and olfactory bulb and is also expressed in superior colliculus, cerebral cortex, hippocampus, striatum, substantia nigra, and hypothalamus (Fig. 2A). By contrast, MALS-2 expression is weak in cerebellum and olfactory bulb but is enriched in cerebral cortex, thalamus, hippocampus, and susbtantia nigra (Fig. 2A). MALS-3 shows the most restricted expression in brain and occurs at highest levels in the cerebellum, olfactory bulb, and superior colliculus and dentate gyrus of hippocampus (Fig. 2A).


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Fig. 2.   Distribution of MALS-1,2,3 in brain. Sagittal sections of mouse brain were stained with isoform-specific MALS antibodies (A) or were hybridized with antisense cRNA probes (B). MALS-1,2,3 proteins and mRNA are discretely localized in distinct brain regions. Regional distributions of protein and mRNA for each MALS isoform are generally correlated such that high levels of MALS-1 are found in cerebellum (CB), cerebral cortex (CTX), dentate gyrus of hippocampus (HP), and olfactory bulb (OB). MALS-2 in enriched in cerebral cortex, substantia nigra (SN), hippocampus, corpus striatum (STR), and thalamus (T). MALS-3 expression is generally diffuse and occurs above background in the cerebellum, superior colliculus, and dentate gyrus of hippocampus. The brain sections used for immunohistochemistry and in situ hybridization are from different sagittal planes; this explains why some structures are labeled by only one technique. For example the inferior olive (IO), which is enriched for MALS-1 and MALS-2 mRNAs, is not contained in the sections used for immunohistochemistry. Pn, Pons.

In situ hybridization for MALS-1,2,3 generally correlates to that seen by immunohistochemistry (Fig. 2B). That is, highest levels for MALS-1 mRNA are found in cerebellar granule cells, cerebral cortex, and dentate gyrus of the hippocampus. MALS-1 mRNA occurs at highest levels in neurons of the inferior olive, which also stain positive for MALS-1 protein (data not shown). MALS-2 hybridization is low in cerebellum but is readily detected in cerebral cortex, hippocampus, pontine nucleus, and inferior olive. MAL-3 mRNA is detected only very weakly throughout the brain but found above background levels in hippocampal dentate gyrus. In a few brain regions there are discrepancies between localization signals detected by immunohistochemistry and in situ hybridization. For example, MALS-2 is enriched in the thalamus by immunohistochemistry but not by in situ hybridization. Also MALS-2 is positive in the pontine nucleus by in situ hybridization but faint by immunohistochemistry. These differences are likely attributable to subcellular sorting of MALS proteins to axons or dendrites.

Higher power micrographs were used to reveal the cellular distribution of the MALS proteins in specific neuronal populations. Throughout the brain, MALS are found predominantly in neurons and occur differentially in neuronal cell bodies and neuropil. In substantia nigra pars compacta (Fig. 3A) and ventral thalamus (Fig. 3E), MALS-1 staining is prominent in neuronal cell bodies. By contrast, MALS-1 is distributed diffusely in the neuropil of cerebral cortex, superior colliculus, and corpus striatum (Fig. 3, B-D). MALS-2 expression is prominent in cell bodies of substantia nigra pars compacta but is also enriched in the neuropil of the substantia nigra pars reticulata (Fig. 3F). In cerebral cortex, MALS-2 intensely labels interneurons (Fig. 3, G and H), which are devoid of MALS-1 or MALS-3. MALS-2 also strongly stains neurons of the pontine tegmental nucleus (Fig. 3I). MALS-3 staining occurs diffusely in neuropil of specific brain regions including the superior colliculus (Fig. 3J), the reticular thalamic nucleus, and the olfactory bulb (Fig. 2, K and L).


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Fig. 3.   Neuronal expression of MALS-1,2,3 in brain. MALS-1 staining was detected in cell bodies of substantia nigra compacta (A), diffuse cortical neuropil (strongest in layer 5; B), diffuse neuropil and superficial gray layer of superior colliculus (C); diffuse neuropil of caudate putamen (D), and cell bodies of ventral thalamic nuclei (E). MALS-2 staining was detected in cell bodies of substantia nigra compacta and diffuse neuropil of substantia nigra reticulata (F), diffuse cortical neuropil and cell bodies of cortical interneron (G), higher magnification of interneurons in neocortex (H), and cell bodies of pontine tegmental nucleus (I). MALS-3 staining was apparent in diffuse neuropil of superior colliculus (J), diffuse neuropil of reticular thalamic nucleus (K), and glomeruli of principal and accessory olfactory bulb (L). Magnifications: B and G, ×50; H, ×400; the rest of panels, ×100.

In hippocampus and cerebellum, MALS proteins are expressed in differential complementary neuronal populations. As shown in Fig. 8A, MALS-1 occurs diffusely in the dendritic fields of hippocampal dentate granule cells. By contrast, MALS-2 is absent from the dentate gyrus of hippocampus. Instead, MALS-2 is abundant in the neuropil of the CA1 region (see Fig. 8C). Also, as found in cerebral cortex, MALS-2 is strikingly concentrated in cell bodies and processes of interneurons. MALS-3 expression in hippocampus is weak but generally resembles that of MALS-1 in the molecular layer of the dentate gyrus (see Fig. 8E).

In cerebellum, MALS-1 is enriched in the cerebellar granule cell layer and is clearly concentrated in the glomeruli. MALS-1 also occurs diffusely in the molecular layer of cerebellum (see Fig. 9A). MALS-2 expression in cerebellum resembles that of MALS-1 but is much weaker. Peroxidase staining for MALS-2 shown in Fig. 9C was intentionally prolonged to bring out the very light labeling. MALS-3 in cerebellum is restricted to the molecular layer (see Fig. 9E).

Targeted Disruption of MALS-1/2-- Because MALS-1,2 are brain-specific and occur in a variety of neuronal populations, we targeted disruption of these genes to help define roles for MALS proteins in neuronal function. For MALS-1 we targeted the second exon, which encodes 39 amino acids (83-201 bp) and is flanked on either side by introns of >10 kb. The MALS-2 gene comprises 6 exons that span 2.3 kb of genomic DNA, so we deleted the entire coding region. Proper targeting of both genes was successful as determined by Southern blotting and PCR analysis (Figs. 4 and 5). For both genes, heterozygous mice were interbred, and both MALS-1 and MALS-2 knockout mice were born at the expected Mendelian frequencies. Also, both MALS-1 and MALS-2 knockout mice are viable, fertile, and indistinguishable from their littermates.


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Fig. 4.   Targeted disruption of MALS-1 gene. A, restriction maps of the wild type murine MALS-1 locus, the targeting construct, and the targeted locus. A 2.2-kb region containing the second exon (119 bp) of MALS-1 gene was replaced by a neomycin cassette (Neo). Filled bars indicate probes used in Southern analysis. Open bars indicate positions of PCR fragments used for genotyping. B, BamHI; E, EcoRI; K, KpnI; N, NotI; X, XbaI; Xh, XhoI. B, Southern blot analysis of genomic DNA from wild type (+/+), heterozygous (+/-), and null (-/-) mice. Using the 5' probe and EcoRI digestion, the wild type and the targeted loci generate 9.5- and 15-kb bands, respectively. Using the 3' probe and BamHI digestion, the wild type and the targeted loci generate 4.5- and 3.0-kb bands, respectively. The neomycin cassette probe recognizes a 15-kb band in the targeted locus after EcoRI digestion. C, PCR genotyping. The wild type and the targeted loci give 367- and 723-bp PCR products, respectively. D, Western blot analysis. Brain homogenate were immunoblotted with the affinity-purified pan-MALS antibody, which recognized all three MALS isoforms. MALS-1 protein is undetectable, but MALS-2 and MALS-3 proteins are detected normally in the MALS-1 null mouse.


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Fig. 5.   Targeted disruption of MALS-2 gene. A, restriction map of the wild type murine MALS-2 locus, the targeting construct, and the targeted locus. A 2.3-kb region spanning six coding exons of MALS-2 gene was replaced by a neomycin cassette (Neo). Exons with the initiation codon (ATG) and the termination codon (Stop) are marked by asterisks. Filled bars indicate probes used in Southern analysis. Open bars indicate positions of PCR fragments used for genotyping. Abbreviations of restriction enzyme sites are as shown in the legend to Fig. 1. B, Southern blot analysis of genomic DNA from wild type (+/+), heterozygous (+/-), and null (-/-) mice. Using the 5' probe and BamHI digestion, the wild type and the targeted loci generate 8.4- and 4.6-kb bands, respectively. Using 3' probe and KpnI digestion, the wild type and the targeted loci generate 20- and 12-kb bands, respectively. The neomycin cassette probe hybridizes to a 12-kb band after KpnI digestion. C, PCR genotyping. The wild type and the targeted loci give 436- and 332-bp products, respectively. D, Western blot analysis. Brain homogenates were immunoblotted with specific antibodies to MALS-1, MALS-2, or MALS-3. MALS-2 protein band is selectively absent in the MALS-2 null mouse.

Western blotting was used to assess MALS protein expression in the knockout mice. Homogenates from whole brain of wild type, MALS-1+/-, and MALS-1-/- were probed with a pan-MALS antibody that reacts with all three isoforms. As shown in Fig. 4D, MALS-1 staining is selectively lost in the MALS-1 knockout. Similarly, immunoblotting extracts from wild type, MALS-2+/-, and MALS-2-/- with isoform specific antibodies shows the expected loss of MALS-2 in the knockouts (Fig. 5D).

To generate double knockouts (DKO), the MALS-1 and MALS-2 mutants were interbred (Fig. 6). F1 progeny, which are heterozygous for each gene, were intercrossed, and 99 pups were analyzed. These F2 pups were born in the predicted Mendelian ratios and included six DKOs (Fig. 6D). As expected, Western blotting of total brain homogenates showed that MALS-1 and MALS-2 are absent from the DKOs. MALS-1/2 DKOs are viable, fertile, and cannot be distinguished from their littermates.


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Fig. 6.   MALS-1/2 double null mice. MALS-1/2 double heterozygous mice were generated by mating female MALS-1-/- mouse (F3 generation on Black Swiss background) and male MALS-2 chimera mouse. MALS-1/2 double null mice were made by intercrossing MALS-1/2 double heterozygous mice. A, PCR genotyping. For genotyping MALS-1 loci, new PCR primers were used for the targeted locus, which give a 189-bp band. The other primers used those shown in the legends to Figs. 4 and 5. An asterisk shows MALS-1/2 double null mouse. B, Southern blot analysis. BamHI-digested genomic DNA was hybridized with the 3' probe and the 5' probe for genotyping MALS-1 loci and MALS-2 loci, respectively. C, Western blot analysis. Brain homogenates were immunoblotted with specific antibodies against MALS-1, MALS-2, or MALS-3. D, genotyping of offspring (99 pups) from parents double heterozygous for MALS-1 and MALS-2. Expected numbers for each genotype are also shown. WT, wild type.

Normal Synaptic Transmission and Plasticity in the MALS-1/2 DKO-- We carried out a series of electrophysiological experiments to examine for possible alterations in excitatory synaptic transmission in the CA1 region of the hippocampus in the MALS-1/2 double knockout mouse. The first set of experiments examined the input-output function of the excitatory synapses using extracellular field potential recording. This was done by varying the stimulus strength and recording the size of the presynaptic fiber volley (input) and comparing it to the size (measured as an initial slope) of the postsynaptic response (fEPSP) (output). A difference in this relationship would indicate a change in the synaptic strength, either because of a change in transmitter release or a change in the responsiveness of the postsynaptic AMPA receptors (AMPARs). As shown in Fig. 7A, the relationship between the fiber volley (input) and the fEPSP slope (output) does not differ between wild type and DKO.


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Fig. 7.   Synaptic transmission in the CA1 region of the hippocampus of the MALS-1 and 2 double knockout is normal. A, a comparison between the size of the fiber volley and the resulting field EPSP measured from seven wild type slices (three animals) and eight knockout slices (four animals). Recordings were categorized according to fiber volley size. B, AMPA/NMDA ratios measured with whole cell recording at +40 mV in the presence and absence of D-APV (100 µM) (seven wild type cells from three animals and six knockout cells from three animals). Average values are 1.08 ± 0.13 and 0.94 ± 0.09 for wild type and knockout, respectively (bottom graph, p = 0.40). Example current traces are shown above the respective bars. C, the graph demonstrates the average fEPSP slope during a 10-min base line and after a tetanic stimulation consisting of three bursts of 100 pulses at 100 Hz separated by 20 s (time of tetanus represented by an arrowhead). A total of six slices from three animals in both wild type and knockout were used in these experiments. Average long term potentiation magnitude 50-60 min after tetanus is 138 ± 14 and 149 ± 16% for wild type and knockout, respectively (p = 0.60). Example field potentials during base line and 50-60 min after tetanus are shown above.

We next addressed whether the DKO might have altered contributions of AMPARs and NMDARs to excitatory synaptic transmission. To examine simultaneously the AMPAR- and NMDAR-mediated components of the EPSC, we performed whole cell recordings and held the cell at +40 mV. The reason for recording at positive membrane potentials is that the Mg block of the NMDAR is removed and the I-V relationship of both the AMPAR and NMDAR components of the EPSC are essentially linear. Responses to activation of both the AMPAR and NMDAR component were recorded first, and then the NMDAR selective antagonist APV was added to completely block the NMDAR component. The AMPAR component was thereby measured directly, and the NMDAR component was obtained by subtracting the AMPAR component from the initial dual component. Fig. 7B shows sample records from a wild type cell (left column), a DKO cell (right column), and a summary graph of results from a number of cells. As shown in the graph, no obvious difference between wild type and DKO is detected. A final set of experiments compared the magnitude of NMDAR-dependent long term potentiation in wild type versus DKO. After obtaining a 10-min base line, a 100 Hz tetanus (1 s repeated three times at an interval of 20 s) was delivered. A large potentiation immediately followed the tetanus that decayed to a stable potentiation of about 50%. No significant difference in the magnitude of the long term potentiation was found between the wild type and DKO.

Molecular Compensation by MALS-3 in Double Knockout Mice-- The absence of an overt phenotype in the MALS-1/2 DKO mutants suggested possible compensation by MALS-3. To assess this possibility we stained brain sections from the DKOs with antibodies to each of the MALS antibodies (Fig. 8). As expected, MALS-1 and MALS-2 staining are abolished in the DKOs (Fig. 8, B and D). Strikingly, MALS-3 expression in the DKOs is up-regulated in the dentate gyrus and the CA-1 layer of hippocampus (Fig. 8F), which normally express MALS-1 and MALS-2, respectively. However, no up-regulation of MALS-3 is seen in hippocampal interneurons, which robustly express MALS-2 in wild type mice. We also assessed possible compensation in cerebellum (Fig. 9). As expected, MALS-1 and MALS-2 staining were lost from cerebellar glomeruli of the DKO mice (Fig. 9, B and D). This loss of MALS-1,2 was compensated by a dramatic up-regulation of MALS-3 in cerebellar glomeruli (Fig. 9F).


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Fig. 8.   Expression of MALS-1, MALS-2, and MALS-3 in hippocampus of wild type and MALS-1/2 double null mice. Immunohistochemical staining of hippocampus from wild type (A, C, and E) and double null (B, D, and F) mice used with specific antibodies to MALS-1 (A and B), MALS-2 (C and D), and MALS-3 (E and F). Note that compensatory changes of MALS-3 expression are evident in the double null mice. Magnification is ×50.


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Fig. 9.   Expression of MALS-1, MALS-2, and MALS-3 in cerebellum of wild type and MALS-1/2 double null mice. Immunohistochemical staining of cerebellum from wild type (A, C, and E) and double null (B, D, and F) mice used specific antibodies to MALS-1 (A and B), MALS-2 (C and D), and MALS-3 (E and F). Magnification is 100×. The inset in F shows higher magnification (400×) of granule cell layer of double null mice stained with MALS-3 antibody. Note the compensatory overexpression of MALS-3 in the glomeruli in the knockout mice.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A primary finding of this study is that MALS proteins occur discretely in specific neuronal populations throughout the brain. This neuronal distribution in mammalian brain contrasts with the localization of LIN-7 to epithelial cells in C. elegans (17). In fact, we did not detect MALS in most non-neuronal cells in brain such as vascular endothelial cells, vascular smooth muscle cells, or epithelial cells. The neuronal distribution for MALS is consistent with the proposed role for these proteins in regulation of synaptic function (9, 11, 12).

We were unable to assign MALS isoforms to specific neurotransmitter systems. For example, MALS-2 occurs in both excitatory granule cells of cerebellum and in inhibitory interneurons of cerebral cortex. In many neuronal populations MALS proteins are differentially expressed such that most neuronal types express only a single MALS isoform. This differential expression is seen clearly in the hippocampus such that MALS-1 occurs uniquely in granule cells of the dentate gyrus, whereas MALS-2 occurs in pyramidal cells of Ammon's Horn and in interneurons (Fig. 8). However, some neurons contain multiple MALS, because both MALS-1 and-2 occur in cerebellar granule cells, and all three MALS isoforms are expressed in the dopaminergic neurons of the substantia nigra pars compacta.

Specific functions for MALS proteins in brain have remained unclear. Biochemical studies have demonstrated that MALS occur in a tight complex with CASK and Mint-1, which are the mammalian homologs of LIN-2 and LIN-10, respectively (9, 11, 18). An N-terminal domain of MALS binds to CASK, which is a membrane-associated guanylate kinase that occurs at cell junctions in association with neurexins and syndecan heparin sulfate proteoglycans (13, 14). CASK is also suggested to translocate to the nucleus and regulate transcription by T-brain-1 (19). Because we do not detect nuclear accumulation of MALS in developing or mature brain, we suspect that MALS do not participate in gene regulation by CASK.

Mint-1 is a neuronal protein that contains PDZ and phosphotyrosine-binding motifs (20). Whereas ligands for the PDZ domain remain unknown, the phosphotyrosine-binding domain of Mint-1 interacts with the cytosolic tail of amyloid precursor protein and slows the processing of amyloid precursor protein to Abeta (21). This role for Mint-1 in processing of transmembrane proteins is consistent with its localization at the Golgi apparatus of developing neurons (11). We do not find MALS staining of Golgi, suggesting that MALS do not assist Mint-1 in early protein processing. In mature brain, Mint-1 occurs in a somatodendritic distribution in specific neurons (11), many of which contain MALS proteins. Coexpression of Mint-1, CASK, and MALS in substantia nigra, olfactory bulb, and layer 5 of cerebral cortex identifies neuronal populations that likely contain a ternary complex of these three PDZ proteins.

The subcellular localization of the CASK/MALS/Mint complex remains uncertain. A presynaptic locus for this complex has been suggested based on the known protein interactions of Mint and CASK. Mint was originally identified via its interaction with Munc-18, a necessary component for synaptic vesicle release (20), and CASK was isolated by interaction with beta -neurexin (22), a neuronal adhesion molecule that is presumed to coat presynaptic terminals. On the other hand, postsynaptic localization for MALS has been suggested based on its fractionation with the PSD and association with PSD-95 and NMDAR (12). Furthermore, the CASK/MALS/Mint complex interacts with a dendritic kinesin motor protein KIF17 and may traffic NMDA containing vesicles to the PSD (23). Immunohistochemical studies reported here are consistent with localization of MALS in both axons and dendrites. This dual subcellular localization is most apparent for MALS-1 in cerebellar granule cells. Immunohistochemical staining shows that MALS-1 protein occurs in granule cells axons in the molecular layer and in postsynaptic glomeruli in the granule cell layer. Future studies to determine definitively the subcellular loci for the CASK/MALS/Mint complex will require dual label immunohistochemistry at the electron micrographic level.

Clues concerning functions for the CASK/MALS/Mint complex may be gained by comparison with C. elegans, in which lin-2/7/10 mutations disrupt normal differentiation of vulval precursor cells (8). This vulvaless phenotype results from failed targeting of the LET-23 tyrosine kinase receptor to the vulval precursor cell basolateral surface, where it must localize to interact with its extracellular ligand. Although the details of LET-23 protein sorting remain uncertain, interaction of the PDZ domain from LIN-7 with the extreme C terminus of LET-23 is a necessary step. By analogy, it seems likely that the PDZ domain of MALS may interact with synaptic receptors (18). Biochemical analysis shows that the PDZ domain of MALS is type I and interacts with proteins terminating with a consensus (E/D)(S/T)X(L/V/F) (12). This sequence is shared by numerous synaptic channels and receptors including glutamate receptors, K+ channel, and Ca2+ channels, all of which have been shown to bind to MALS (12).

To determine essential roles for MALS proteins in synaptic assembly, we targeted disruption of MALS-1 and-2 because they are highly enriched in brain. Our electrophysiological analysis of excitatory synaptic transmission in the CA1 region of the hippocampus failed to reveal any clear effects of deleting both MALS-1 and 2 on synaptic physiology. The comparison of the size of presynaptic fiber volley to the size of the postsynaptic response (input/output curve) provides the best, albeit rather crude, measure for the strength of basal synaptic transmission. Thus, any change in the ability of the synapse to release transmitter should show up as a change in this measurement. In addition, any change in the ability of the AMPARs to respond to released glutamate should alter this measurement. The lack of change in the input/output curve does not exclude the possibility that defects exist. For instance, the ability of the synapse to respond to repetitive stimulation involves a number of processes that might not be detected by examining the synaptic responses at low frequency stimulation. The analysis of the relative contribution of the AMPARs and NMDARs to the EPSC indicates whether there has been any differential effect on the function of these two receptors. For instance, if MALS were to selectively effect the trafficking of one subtype of receptor (23), this would be detected as a change in the AMPA/NMDA ratio. This measurement provides no information about the absolute strength of transmission because the size of the responses is dictated by the number of presynaptic fibers activated by the stimulating electrode. In a final set of experiments we examined whether the double KO might effect the magnitude of NMDAR dependent long term potentiation. Again, we were unable to detect any obvious difference between the wild type and double KO mouse.

Although we failed to identify physiological abnormalities in the MALS-1/2 double knockouts, we detected striking up-regulation of MALS-3 in the mutants. This up-regulation was apparent in numerous brain regions but was not universal. For example, MALS-3 was not induced in interneurons, which normally express MALS-2, implying that regulation of MALS proteins is different in excitatory and inhibitory neurons. Knockout of other PDZ proteins, such as PSD-95 or PSD-93 knockouts do not cause up-regulation of paralogous proteins, suggesting that dynamic expression may be particularly important for the MALS protein family (24, 25). Because MALS-3 may functionally compensate for the lack of MALS-1/2, it will now be important to develop triple knockouts to help define the essential functions for this family of PDZ proteins.

    ACKNOWLEDGEMENTS

We thank Drs. Juanito Meneses and Roger Pedersen in the UCSF mutagenesis core for helping generate the transgenic mice.

    FOOTNOTES

* This work was supported by grants from the National Institutes of Health (to D. S. B. and R. A. N.), the Howard Hughes Medical Institute Research Resources Program and the Human Frontier Research Program (to D. S. B.), and Bristol-Myers Squibb (to R. A. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a fellowship grant from the Human Frontier Science Program. Present address: Dept. of Neurology, Tokyo Metropolitan Inst. for Neuroscience 2-6, Musashidai, Fuchu City, Tokyo 183-8526, Japan.

|| Member of the Keck center for Integrative Neuroscience and the Silvo Conte Center for Neuroscience Research.

** Established investigator for the American Heart Association. To whom correspondence should be addressed: University of California at San Francisco School of Medicine, 513 Parnassus Ave., San Francisco, CA 94143-0444. Tel.: 415-476-6310; Fax: 415-476-4929; E-mail: bredt@itsa.ucsf.edu.

Published, JBC Papers in Press, December 4, 2000, DOI 10.1074/jbc.M009334200

    ABBREVIATIONS

The abbreviations used are: PSD, postsynaptic density; AMPA, alpha -amino-3-hydroxyl-5-methyl-4-isoxazolepropionate; AMPAR, AMPA receptor; DKO, double knockout; EPSP, excitatory postsynaptic potential; NMDA, N-methyl-D-aspartate; NMDAR, NMDA receptor; bp, base pair(s); PCR, polymerase chain reaction; kb, kilobase(s).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Hsueh, Y. P., and Sheng, M. (1998) Prog. Brain Res. 116, 123-131[Medline] [Order article via Infotrieve]
2. Kennedy, M. B. (1998) Brain Res. Rev. 26, 243-257[CrossRef][Medline] [Order article via Infotrieve]
3. Craven, S. E., and Bredt, D. S. (1998) Cell 93, 495-498[Medline] [Order article via Infotrieve]
4. Doyle, D. A., Lee, A., Lewis, J., Kim, E., Sheng, M., and MacKinnon, R. (1996) Cell 85, 1067-1076[Medline] [Order article via Infotrieve]
5. Hillier, B. J., Christopherson, K. S., Prehoda, K. E., Bredt, D. S., and Lim, W. A. (1999) Science 284, 812-815[Abstract/Free Full Text]
6. Woods, D. F., and Bryant, P. J. (1991) Cell 66, 451-464[Medline] [Order article via Infotrieve]
7. Budnik, V., Koh, Y. H., Guan, B., Hartmann, B., Hough, C., Woods, D., and Gorczyca, M. (1996) Neuron 17, 627-640[Medline] [Order article via Infotrieve]
8. Kaech, S. M., Whitfield, C. W., and Kim, S. K. (1998) Cell 94, 761-771[Medline] [Order article via Infotrieve]
9. Butz, S., Okamoto, M., and Südhof, T. C. (1998) Cell 94, 773-782[Medline] [Order article via Infotrieve]
10. Rongo, C., Whitfield, C. W., Rodal, A., Kim, S. K., and Kaplan, J. M. (1998) Cell 94, 751-759[Medline] [Order article via Infotrieve]
11. Borg, J.-P., Lõpez-Figueroa, M. O., de Taddèo-Borg, M., Kroon, D. E., Turner, R. S., Watson, S. J., and Margolis, B. (1999) J. Neurosci. 19, 1307-1316[Abstract/Free Full Text]
12. Jo, K., Derin, R., Li, M., and Bredt, D. S. (1999) J. Neurosci. 19, 4189-4199[Abstract/Free Full Text]
13. Cohen, A. R., Woods, D. F., Marfatia, S. M., Walther, Z., Chishti, A. H., Anderson, J. M., and Wood, D. F. (1998) J. Cell Biol. 142, 129-138[Abstract/Free Full Text]
14. Hsueh, Y. P., Yang, F. C., Kharazia, V., Naisbitt, S., Cohen, A. R., Weinberg, R. J., and Sheng, M. (1998) J. Cell Biol. 142, 139-151[Abstract/Free Full Text]
15. Nehls, M., Kyewski, B., Messerle, M., Waldschütz, R., Schüddekopf, K., Smith, A. J., and Boehm, T. (1996) Science 272, 886-889[Abstract]
16. Sassoon, D., and Rosenthal, N. (1993) Methods Enzymol. 225, 384-404[Medline] [Order article via Infotrieve]
17. Simske, J. S., Kaech, S. M., Harp, S. A., and Kim, S. K. (1996) Cell 85, 195-204[Medline] [Order article via Infotrieve]
18. Bredt, D. S. (1998) Cell 94, 691-694[Medline] [Order article via Infotrieve]
19. Hsueh, Y. P., Wang, T. F., Yang, F. C., and Sheng, M. (2000) Nature 404, 298-302[CrossRef][Medline] [Order article via Infotrieve]
20. Okamoto, M., and Südhof, T. C. (1997) J. Biol. Chem. 272, 31459-31464[Abstract/Free Full Text]
21. Borg, J. P., Yang, Y., De Taddéo-Borg, M., Margolis, B., and Turner, R. S. (1998) J. Biol. Chem. 273, 14761-14766[Abstract/Free Full Text]
22. Hata, Y., Butz, S., and Südhof, T. C. (1996) J. Neurosci. 16, 2488-2494[Abstract]
23. Setou, M., Nakagawa, T., Seog, D.-H., and Hirokawa, N. (2000) Science 288, 1796-1802[Abstract/Free Full Text]
24. Migaud, M., Charlesworth, P., Dempster, M., Webster, L. C., Watabe, A. M., Makhinson, M., He, Y., Ramsay, M. F., Morris, R. G., Morrison, J. H., O'Dell, T. J., and Grant, S. G. (1998) Nature 396, 433-439[CrossRef][Medline] [Order article via Infotrieve]
25. McGee, A. W., Topinka, J. R., Hashimoto, K., Petralia, R. S., Kakizawa, S., Kauer, F., Wenthold, R. J., Kano, M., and Bredt, D. S. J. Neurosci. (2001), in press.


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.