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The Journal of Neuroscience, July 21, 2004, 24(29):6629-6637; doi:10.1523/JNEUROSCI.1610-04.2004
Cellular/Molecular
A Complete Genetic Analysis of Neuronal Rab3 Function
Oliver M. Schlüter,1,2,3 Frank Schmitz,4 Reinhard Jahn,3 Christian Rosenmund,3 andThomas C. Südhof1 1Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9111, Max Planck Institutes for 2Experimental Medicine and 3Biophysical Chemistry, 370701 Göttingen, Germany, and 4Department of Anatomy, Universität des Saarlandes, 66123 Saarbrücken, Germany
Abstract
Top
Abstract
Introduction
Rab3A, Rab3B, Rab3C, and Rab3D are closely related GTP-binding proteins of synaptic vesicles that may function in neurotransmitter release. We have produced knock-out (KO) mice for Rab3B and Rab3C and crossed them with previously generated Rab3A and 3D knock-out mice to generate double, triple, and quadruple Rab3 knock-out mice. We have found that all single and double Rab3 knock-out mice are viable and fertile. Most triple Rab3 knock-out mice perish whenever Rab3A is one of the three deleted proteins, whereas all triple knock-out mice that express Rab3A are viable and fertile. Finally, all quadruple knock-out mice die shortly after birth. Quadruple Rab3 KO mice initially develop normally and are born alive but succumb to respiratory failure. Rab3-deficient mice display no apparent changes in synapse structure or brain composition except for a loss of rabphilin, a Rab3-binding protein. Analysis of cultured hippocampal neurons from quadruple knock-out mice uncovered no significant change in spontaneous or sucrose-evoked release but an30% decrease in evoked responses. This decrease was caused by a decline in the synaptic and the vesicular release probabilities, suggesting that Rab3 proteins are essential for the normal regulation of Ca2+-triggered synaptic vesicle exocytosis but not for synaptic vesicle exocytosis as such. Our data show that Rab3 is required for survival in mice and that the four Rab3 proteins are functionally redundant in this role. Furthermore, our data demonstrate that Rab3 is not in itself essential for synaptic membrane traffic but functions to modulate the basic release machinery.
Key words: membrane fusion; synaptic transmission; GTP-binding proteins; neurotransmitter release; RIM; synaptic vesicles
Introduction
Rabs are GTP-binding proteins that are generally involved in regulating membrane traffic (for review, see Darchen and Goud, 2000; Tuvim et al., 2001
Many, occasionally divergent, functions in exocytosis have been proposed for Rab3 proteins. These functions range from fusion to docking and to the biogenesis of vesicles (for review, see Darchen and Goud, 2000
No KOs in multiple Rab3 proteins (or in any other Rab protein) have been analyzed, and it is conceivable that compensation by Rab3B and/or Rab3C confounds the phenotype of Rab3A and Rab3D KO mice. To address this possibility, we have generated and analyzed mice that lack all Rab3 isoforms. Our data demonstrate that Rab3 is essential for mouse survival and that Rab3 isoforms are functionally redundant. However, our data show that Rab3 is not in itself required for exocytosis. In recurrent synapses (autapses) formed by cultured hippocampal neurons, deletion of Rab3 induces a discrete reduction in the neurotransmitter release probability without changes in other basic parameters of synaptic vesicle exocytosis. These data suggest that in the brain, Rab3 proteins function as modulators of neurotransmitter release that are not involved in the fundamental machinery of docking and fusion of synaptic vesicles but act as modulators of the release machinery.
Materials and Methods
Genomic cloning. Using a probe from the 5' end of the rat Rab3C cDNA derived by PCR [primers 337 (GCGGATCCCATATGAGACACGAAGCTCCC) and 338 (GCGGATCCAATCTGAAGCTTGATTCT)], we isolated 22 clones from aFixII 129SV/J genomic library (Stratagene, La Jolla, CA) and characterized these clones by restriction enzyme mapping and sequencing (Südhof, 1990
Generation and maintenance of Rab3 KO mice. To delete the homologous 5' exon of the Rab3B and Rab3C genes, we constructed similar targeting vectors (see Fig. 1). We cloned an
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Figure 1. Generation of Rab3B and Rab3C knock-out mice. The diagrams depict the structure of the Rab3B and Rab3C wild-type gene (Gene) and the structure of the targeting vector used for homologous recombination (Targeting Vector). Positions of strategic restriction enzyme sites are indicated, and the residue numbers encoded by the exons (represented as black boxes in the gene structures) are listed below the exons. Localizations of outside probes (OP) and PCR primers (arrows with numbers of letters) are also shown. TK, Thymidine kinase.
Antibodies. The antibodies used in this study have been described previously (Rosahl et al., 1995Spirometry. Within 1 hr after birth, mice were placed in a closed recording chamber of defined volume. Pressure changes were detected using a high-sensitivity pressure transducer, and the signals were amplified and digitized using standard acquisition software. Breathing activity was recorded for three consecutive trials at 37°C for a total duration of 5 min.
Cell culture and electrophysiology. E19 mouse embryos of Rab3A+/- or Rab3A+/-BCD-/- timed matings were delivered by cesarean section. Hippocampi from the entire litter were isolated and cultured in chemically defined media (NBA; Invitrogen, San Diego, CA) supplemented with B27 (Invitrogen) on glial microdot islands (Bekkers and Stevens, 1991
) were filled with internal solution containing 125 mM K-gluconate, 10 mM NaCl, 4.6 mM MgCl2,4mM ATP-Na2, 0.3 mM GTP-Na2,15mM creatine phosphate, 20 U/ml phosphocreatine kinase, and 1 mM EGTA, pH 7.3, also adjusted to 300 mOsm. Solutions were applied using a fast-flow application system (Pyott and Rosenmund, 2002
Data analysis. Spontaneous EPSCs were monitored for 1-2 min and analyzed with a template detection program (Axograph 4.8; Axon Instruments). The threshold for detection was set to 3.5 times the baseline SD. Captured spontaneous EPSCs of individual cells were averaged to determine amplitude, charge, and decay constant. The vesicular release probability was calculated by dividing the charge of an evoked response by the charge of the burst component of the response to hypertonic sucrose solution of the same cell. Vesicle release time course was determined by deconvolving the EPSC time course with the mean spontaneous EPSC time course using a custom-written software module within Axograph 4.6 (gift from Dr. John Clements, Australian National University, Canberra, Australia). Statistical analyses were performed using Student's t test, and p < 0.05 was considered significant. For data fitted with exponential curves, the goodness of fit for each curve fitting was determined by comparing both the
2 and sum-of-squares errors.
Miscellaneous procedures. Light and electron microscopy were performed on neurons cultured at high density on a confluent glia feeder layer that were fixed after 12 d in culture with 4% paraformaldehyde and 1% glutaraldehyde in PBS. Electron microscopy, tissue homogenization, immunoblotting, and protein quantifications using 125I-labeled secondary antibodies and radioactivity measurements on immunoblots were performed as described previously (Rosahl et al., 1995
Results
Generation of KO mice lacking all four Rab3 isoforms
We produced Rab3B and Rab3C KO mice using the strategy that we previously applied for the generation of Rab3A and Rab3D KO mice (Geppert et al., 1994Preliminary analyses indicated that all single and double KO mice and at least some of the triple KO mice were viable and fertile (see systematic analysis below). Therefore, we first aimed to confirm that the KO strategy effectively deleted the various Rab3 proteins. For this purpose, we analyzed all four combinations of triple Rab3 KO mice by immunoblotting with isoform-specific antibodies. We examined brain samples with Rab3A-, Rab3B-, and Rab3C-specific antibodies and pituitary samples with Rab3D-specific antibodies. Pituitary samples were chosen for the Rab3D analysis because Rab3D levels are higher in pituitary than in brain and the Rab3D antibodies exhibit a relatively low affinity, making it difficult to measure Rab3D levels in brain (Schlüter et al., 2002
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Figure 2. Immunoblotting analysis of adult triple Rab3 KO mice. Brain (for Rab3A, Rab3B, and Rab3C) or pituitary (for Rab3D) homogenates from young-adult (6- to 8-week-old) triple KO mice that express only one of the four Rab3 isoforms were examined by immunoblotting. WT, Wild type.
Rab3-deficient mice die at birth
To determine whether deletions of various Rab3 proteins alter the survival of mice, we performed systematic breeding experiments. Surprisingly, in view of the many functions associated with Rab3 in previous studies (Francis et al., 2002
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Figure 3. Survival analysis of Rab3 KO mice. Mice that were homozygous mutants for two Rab3 isoforms and heterozygous for the remaining two Rab3 isoforms were mated, and the genotypes of surviving adult offspring were determined. The frequency of various genotypes is plotted as percentage of total (gray background indicates the expected frequency based on Mendelian inheritance; asterisks indicate genotypes that exhibit impaired survival). Bottom, Plus signs refer to homozygous wild type, asterisks refer to heterozygous, and minus signs refer to homozygous KO.
We detected no surviving quadruple Rab3 KO (ABCD-/-) mice. For all combinations of triple KO mice, at least some mice survived. Triple KO mice that lack Rab3B, Rab3C, and Rab3D (BCD-/- mice) were fully viable even when the remaining Rab3A gene was a heterozygous mutant. Thus, the four Rab3 isoforms together are essential for survival, but a single wild-type Rab3A allele is sufficient to reverse the lethality of the quadruple Rab3 KO (Fig. 3). In contrast, a heterozygous wild-type Rab3C or Rab3B allele rescued survival of the quadruple KO only byRab3 KO mice exhibit respiratory failure
Quadruple Rab3 KO mice were born alive and initially reacted to stimuli. However, quadruple KO mice were distressed, lacked stomach milk, and suffered from cyanosis (Fig. 4A). The quadruple KO mice died soon after birth but survived longer when placed in a high-oxygen atmosphere (95% O2-5% CO2), indicating that the mice perished because of impaired breathing. Consistent with this hypothesis, whole-body plethysmography showed that newborn quadruple KO mice exhibited a shallow and irregular breathing pattern (Fig. 4 B). The lungs, however, appeared to be fully inflated as judged by the fact that lungs from newborn quadruple KO mice floated on water (data not shown). Triple KO mice lacking Rab3A displayed a respiratory impairment that was similar to but less severe than that seen in the quadruple KO mice. Triple KO mice that survived despite the respiratory problems during the perinatal period exhibited abnormal cage behavior and were unable to mate. Weight analyses indicated that all triple KO mice in which Rab3A was deleted together with two of the remaining three Rab3 isoforms exhibited a decrease in body weight at 5 weeks of age (Fig. 5). In contrast, even a single wild-type allele of Rab3A not only conferred full survival to triple Rab3 KO mice that lacked all other Rab3 isoforms (Fig. 3) but also rescued the deficit in mating and weight (Fig. 5 and data not shown).
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Figure 4. Rab3 quadruple KO mice perish from respiratory failure. A, Pictures of triple (Rab3A+/+-BCD-/-) and quadruple Rab3 KO mice (Rab3ABCD-/-) at postnatal day 1. Note the lack of milk and cyanosis in the quadruple KO mice. B, Respiratory activity measured in Rab3A+/+-BCD-/- triple (left) and ABCD-/- quadruple (right) KO mice at birth using whole-body plethysmography.
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Figure 5. Body weights of surviving Rab3 triple KO mice. Weights of male and female 5-week-old mice that are triple KO for three Rab3 isoforms and heterozygous or wild type for the fourth. An asterisk indicates a statistically significant difference from Rab3A+/--BCD-/- or Rab3A+/+-BCD-/- at p < 0.05; the number of individual mice is given at the bottom of each bar. Bottom, Plus signs refer to homozygous wild type, asterisks refer to heterozygous, and minus signs refer to homozygous KO.
Brain composition of Rab3 KO mice
We next asked whether deletion of Rab3 proteins caused a major alteration in brain structure or composition. We uncovered no striking abnormalities by microscopy (data not shown). To detect potential changes in brain composition, we quantitated the levels of 26 neuronal proteins in newborn Rab3 quadruple KO mice using littermate triple KO mice expressing Rab3A as a control, because these triple KO mice exhibit no change in weight, fertility, or survival. We detected no significant change in any protein except for rabphilin (Table 1). Rabphilin is a synaptic vesicle protein and putative Rab3 effector that requires Rab3 for binding to synaptic vesicles (Li et al., 1994(Wang et al., 1997
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Table 1. Protein levels in total brain homogenates of postnatal day 1 mice
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Figure 6. Rabphilin levels in brains from Rab3 KO mice. A, Immunoblots of rabphilin in brains from the indicated adult triple KO mice. B, Proteins levels were quantified in adult mice by immunoblotting using 125I-labeled secondary antibodies with phosphorimager detection and are plotted as percentage of the wild-type control. See Table 1 for quantifications of protein levels in newborn quadruple KO mice. WT, Wild type.
Spontaneous neurotransmitter release in Rab3 knock-out mice
To examine the properties of synapses from Rab3 KO mice, we cultured hippocampal neurons from fetal mice (embryonic day 19) on microislands of astrocytes, conditions that induce formation of autapses (Bekkers and Stevens, 1991Immunofluorescence analysis showed that synapses were formed at a similar density in cultured neurons from various wild-type and Rab3 KO mice (data not shown). These synapses, as viewed by electron microscopy, exhibited typical features with abundant presynaptic vesicles, including vesicles that are docked at the active zone (Fig. 7). Using whole-cell recordings, we found that the frequency of spontaneous release events and their properties (e.g., amplitudes, rise times, duration, and decay kinetics) were indistinguishable between quadruple KO neurons that lack all Rab3 isoforms and control triple KO neurons that lack Rab3A (Fig. 8). Thus, deletion of Rab3 does not cause major alterations in synapse formation or synaptic properties such as the size of synaptic vesicles, filling of synaptic vesicles with neurotransmitters, number of postsynaptic receptors, or kinetics of neurotransmitter release.
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Figure 7. Structure of synapses formed by cultured neurons from Rab3 KO mice. Electron micrographs of synapses formed in cultured hippocampal neurons from quadruple Rab3ABCD-/- KO mice (a, c, e, g) and triple Rab3BCD-/- (b, d, f, g). pr, Presynaptic compartments; po, postsynaptic compartments; m, mitochondria. Arrows in g and h point to docked vesicles in a high-magnification view of the synapse. Scale bars: a-d, 300 nm; e, f, 200 nm; g, h, 80 nm.
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Figure 8. Spontaneous synaptic events in Rab3 KO mice. Representative traces of spontaneous synaptic events (top) and summary of the properties of spontaneous events (bottom) recorded in neurons from triple Rab3BCD-/- and quadruple Rab3ABCD-/- KO mice. Recordings were performed in cultured hippocampal neurons that had formed autapses (n = 39 for both genotypes).
Evoked neurotransmitter release in Rab3 knock-out mice
We next examined whether synaptic responses evoked by presynaptic action potentials are altered in Rab3-deficient synapses. We found that the size of the EPSC induced by action potentials wasfast, 1.7 ± 0.3 msec,
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Figure 9. Evoked synaptic responses in Rab3 KO mice. A, Representative traces of evoked EPSCs triggered by somatic depolarization (arrow, from -70 to 0 mV for 2 msec). Stimulation current was blanked for display purposes. B, Mean cumulative vesicle release from triple (BCD-/-; n = 124) and quadruple (ABCD-/-; n = 165) KO neurons. Error bars indicate SE (p < 0.01). The quantal content and vesicular release time course were calculated by deconvolving synaptic responses with the mean spontaneous synaptic response. The time course of release was determined from a subset of these data (BCD-/-, n = 27; ABCD-/-, n = 29) by fitting the curves to a two-exponential function and was not significantly different between the two genotypes. The dashed lines represent baseline current (A) and baseline release rate (B), respectively.
Is the decrease in evoked responses in the quadruple KO neurons attributable to a decrease in the number of release-ready vesicles or to an impairment in Ca2+ triggering of release? To distinguish between these possibilities, we measured the readily releasable pool of synaptic vesicles. We recorded synaptic responses to hypertonic sucrose (Fig. 10A) that acts by an unknown, Ca2+-independent mechanism to trigger exocytosis of primed vesicles (Rosenmund and Stevens, 1996
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Figure 10. Response to sucrose stimulation in Rab3-deficient synapses. A, Representative traces of synaptic responses to hypertonic sucrose. The dashed lines indicate baseline. B, Average size of sucrose-evoked synaptic responses (n = 141 and 165 for BCD-/- and ABCD-/- neurons, respectively). The decrease in the quadruple KO synapses is not statistically significant (p = 0.16).
Deletion of Rab3 decreases the synaptic and the vesicular release probability
We next asked whether the release probability is decreased in Rab3-deficient neurons. To test this, we evaluated the vesicular release probability (the probability of a given vesicle to fuse when triggered by an action potential) as the ratio of vesicles released by an action potential to the vesicles released by hypertonic sucrose. As shown in the cumulative probability plot (Fig. 11A), the distribution of release probabilities as measured from 141 individual triple deficient and 165 individual quadruple KO neurons covered a wide range of release probabilities, yet shifted uniformly to lower probabilities in the quadruple KO.
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Figure 11. Decreased release probability in Rab3 KO neurons. A, Cumulative plot of the vesicular release probability. Individual values were calculated as the ratio of the synaptic charge of action potential-induced to sucrose-induced EPSCs for individual neurons. B, Stimulus-dependent progressive block of EPSCs with the irreversible NMDA-type glutamate receptor antagonist MK-801. The normalized NMDA receptor-dependent component of the EPSCs was plotted (see Materials and Methods for details). Synaptic responses were evoked at 0.33 Hz (n = 13 cells of 3 cultures). Inset, Transformation of the data by expanding the time axis of the Rab3BCD-/- 1.8-fold causes complete superposition of the two curves.
Using an independent approach to measure release probability, we also monitored NMDA receptor-mediated synaptic responses to low-frequency stimulation (0.33 Hz) in the presence of MK-801. MK-801 is an irreversible NMDA receptor antagonist that only blocks activated receptors; thus, the progressive block rate of EPSCs by MK-801 during ongoing stimulation is a direct measure of the synaptic release probability (Hessler et al., 1993
Discussion
Rab3 proteins are abundant GTP-binding proteins of synaptic vesicles and other secretory organelles. Mammals express four differentially distributed Rab3 isoforms: Rab3A, Rab3B, Rab3C, and Rab3D. Although a large number of experiments using in vitro approaches tie Rab3 proteins to many different functions (Chung et al., 1999We next examined why the Rab3 KO mice die. The major cause of death appeared to be irregular respiratory activity, although other factors such as dehydration may have contributed. Irregular respiratory activity points to a defect of the respiratory circuits in the brainstem. This, together with the preferential localization of Rab3 proteins to synaptic vesicles, indicates a possible impairment in synaptic neurotransmitter release. To study this, we examined the structure and composition of the brain and used cultured hippocampal neurons to test synaptic function. We detected no obvious developmental abnormalities, changes in synapse structure, or alterations in brain composition (Figs. 6, 7; Table 1). Although this does not exclude a subtle developmental effect of the Rab3 KO, this observation argues against a major developmental aberration.
In a final set of experiments, we studied synaptic transmission in the quadruple KO mice. Surprisingly, we found that synaptic vesicle exocytosis exhibited a discrete impairment of release that manifests as an
How does a 30% decrease in release translate into respiratory failure and a 100% lethality in quadruple Rab3 KO mice? At least two hypotheses that are not mutually exclusive can be advanced to explain the origin of the respiratory failure in the Rab3 KO mice: (1) In the present study, our experiments were restricted to measuring excitatory responses in cultured hippocampal neurons elicited by low-frequency stimulation. However, readouts of synaptic networks depend on the precise synaptic properties of the neurons in situ and the balance between excitation and inhibition. Considering the fact that the release properties of synapses are very heterogeneous and that our data indicate a selective role in shaping release properties, different synapse types may be regulated differentially by Rab3 proteins. This possibility is supported by the divergent effects of the Rab3A deletion on two different excitatory synapses in the hippocampus, Schaffer collateral-commissural fiber synapses (Geppert et al., 1994
Previous studies have examined the phenotype of mice lacking the Rab3 GDP-GTP exchange factor (Rab3-GEF) (Yamaguchi et al., 2002
Different from the Rab3-GEF knock-out mice, knock-out mice for the Rab3 effectors rabphilin and RIM1
Our data thus show that Rab3 joins the ranks of proteins that are present at high concentration at the synapse but primarily perform a regulatory function, similar to synapsins, SV2 (synaptic vesicle protein 2), and other synaptic vesicle proteins. The selective requirement for Rab3 in modulating fusion uncovered here is unusual for a Rab protein. The requirement suggests that the synapse may have exploited a protein family normally used in a number of basic membrane trafficking functions (e.g., docking, transport, and budding) for a specialized role. Among others, this indicates that other Rab proteins likely play central roles in synaptic vesicle exocytosis. Identification of these Rabs forms one of the major challenges for the future.
Footnotes
Received April 27, 2004; revised May 31, 2004; accepted June 2, 2004.We thank Dr. John Clements for providing a software module in Axograph 4 to deconvolve vesicular release time course; Dr. M. Geppert, Dr. N. Brose, and T. Jo for advice in the early stages of this project; and Dr. H. Riedesel, F. Benseler, Y. Thannhäuser, I. Leznicki, and A. Roth for technical support.
Correspondence should be addressed to Thomas C. Südhof, Center for Basic Neuroscience, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, NA4.118, Dallas, TX 75390-9111. E-mail: Thomas.Sudhof{at}UTSouthwestern.edu.
O. M. Schlüter's present address: Department of Psychiatry, Stanford University, 1201 Welch Road, Room P152, Stanford, CA 94305.
C. Rosenmund's present address: Department of Neuroscience and Molecular and Human Genetics, Baylor College of Medicine, Room 833E, One Baylor Plaza, Houston, TX 77030.
Copyright © 2004 Society for Neuroscience 0270-6474/04/246629-09$15.00/0
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