From the Departments of 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
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ABSTRACT |
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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.
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.
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 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 M 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.
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).
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).
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.
Western blotting was used to assess MALS protein expression in the
knockout mice. Homogenates from whole brain of wild type, MALS-1+/
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.
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.
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).
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 A 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 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
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.
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
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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.
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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.
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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.
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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.
, 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).
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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.
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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.
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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
(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.
-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.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Juanito Meneses and Roger Pedersen in the UCSF mutagenesis core for helping generate the transgenic mice.
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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
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ABBREVIATIONS |
---|
The abbreviations used are:
PSD, postsynaptic
density;
AMPA, -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).
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REFERENCES |
---|
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---|
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 |
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 |
12. |
Jo, K.,
Derin, R.,
Li, M.,
and Bredt, D. S.
(1999)
J. Neurosci.
19,
4189-4199 |
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 |
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 |
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 |
21. |
Borg, J. P.,
Yang, Y.,
De Taddéo-Borg, M.,
Margolis, B.,
and Turner, R. S.
(1998)
J. Biol. Chem.
273,
14761-14766 |
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 |
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. |