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Double-knockout mice for  - and  -synucleins: Effect on synaptic functions

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Double-knockout mice for  - and  -synucleins: Effect on synaptic functions
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  Double-knockout mice for   - and   -synucleins:Effect on synaptic functions Sreeganga Chandra*, Francesco Fornai † , Hyung-Bae Kwon ‡ , Umar Yazdani § , Deniz Atasoy*, Xinran Liu*,Robert E. Hammer ¶ , Giuseppe Battaglia † , Dwight C. German § , Pablo E. Castillo ‡ , and Thomas C. Su ¨ dhof*  ** †† *Center for Basic Neuroscience, Departments of   Molecular Genetics,  § Psychiatry, and  ¶ Biochemistry, and **Howard Hughes Medical Institute, University ofTexas Southwestern Medical Center, Dallas, TX 75390;  † Department of Human Morphology and Applied Biology, University of Pisa and Istituto di Ricovero eCura a Carattere Scientifico, Istituto Neurologico Mediterraneo Neuromed, 86077 Pozzilli, Italy; and  ‡ Department of Neuroscience, Albert Einstein Collegeof Medicine, Bronx, NY 10461Contributed by Thomas C. Su¨dhof, September 2, 2004 Anabundantpresynapticprotein,  -synuclein,iscentrallyinvolvedin the pathogenesis of Parkinson’s disease. However, conflictingdata exist about the normal function of   -synuclein, possiblybecause  -synucleinisredundantwiththeverysimilar  -synuclein.To investigate the functions of synucleins systematically, we havenow generated single- and double-knockout (KO) mice that lack  -and  or   -synuclein. We find that deletion of synucleins in micedoes not impair basic brain functions or survival. We detected nosignificant changes in the ultrastructure of synuclein-deficientsynapses, in short- or long-term synaptic plasticity, or in the poolsize or replenishment of recycling synaptic vesicles. However,protein quantitations revealed that KO of synucleins caused selec-tive changes in two small synaptic signaling proteins, complexinsand 14-3-3 proteins. Moreover, we found that dopamine levels inthebrainsofdouble-KObutnotsingle-KOmiceweredecreasedby  20%. In contrast, serotonin levels were unchanged, and dopa-mine uptake and release from isolated nerve terminals werenormal. These results show that synucleins are not essentialcomponents of the basic machinery for neurotransmitter releasebut may contribute to the long-term regulation and  or mainte-nance of presynaptic function. T he abundant soluble protein  -synuclein is highly enriched innerve terminals (1–4) and has a central role in the patho-genesis of Parkinson’s disease (5, 6). Pathologically, Parkinson’sdisease is characterized by Lewy bodies, which are eosinophilicinclusions in the neuronal cytoplasm that contain   -synucleinfilaments (7). Genetically, a subset of familial Parkinson’s dis-ease patients harbors a mutant  -synuclein gene (6, 8–10). Thus,  -synuclein is linked to Parkinson’s disease both genetically andneuropathologically. Although the role of   -synuclein in Parkinson’s disease is wellestablished, its normal function remains unclear. Synuclein isabsent from invertebrates, suggesting that it is not essential forsynaptic transmission but may be involved in a vertebrate-specific specialization of synapses (e.g., synaptic plasticity). Thisconclusion is supported by the relatively late translocation of   -synuclein into presynaptic terminals during synaptogenesis,after functional synapses have been established (11). However,investigation of synapses lacking   -synuclein led to conflictingresults. Whereas the srcinal characterization of   -synuclein KOmice failed to detect major changes in the structure and functionof synapses [only a minor use-dependent alteration of dopaminerelease was observed (12)], subsequent studies indicated thatdeletion of   -synuclein induces a massive loss of synaptic vesiclesfrom nerve terminals, a decrease in synaptic responses duringrepetitive stimulation, and an impairment in the recovery of synaptic responses from use-dependent depression (13, 14).These studies have led to the notion that, consistent with itslocalization,   -synuclein may regulate synaptic plasticity (re- viewed in ref. 15). A confounding problem in studying   -synuclein is the pres-ence of    - and    -synucleins, two highly homologous isoforms(1–3, 16–20) (reviewed in ref. 21). The   - and   -synucleins are widely colocalized in presynaptic nerve terminals throughout thebrain but absent from peripheral tissues. In contrast,   -synucleinis largely absent from forebrain (13, 19) but is abundant inspecialized neurons, such as dorsal root ganglia (17). It is alsopresent in nonneuronal tissues (19, 20) and overexpressed inbreast cancer cells (18). The high degree of coexpression of    -and   -synuclein indicates the potential for functional redun-dancy, which may have obscured an essential role of    -synucleinin  -synuclein KO mice. To address this possibility, we have nowgenerated single- and double-KO mice of    - and   -synucleins.Our data demonstrate that the synucleins are not essential forbasic synaptic functions (e.g., neurotransmitter release, synaptic vesicle numbers and pools, and synaptic plasticity) but areredundantly required for maintaining normal dopamine levels inthe nigrostriatal system. Materials and Methods General.  All data are given as mean    SEM (except for ultra-structural data, which are given as mean  SD), and all tests forstatistical significance were performed with Student’s  t  test. Generation of   -Synuclein KO Mice.  By using genomic clones con-taining the entire   -synuclein gene, we constructed a targeting vector in which the first coding exon is flanked by loxP sites (Fig.1). The vector was used in R1 embryonic stem cells (22) forstandard homologous recombination experiments (23). The result-ing mutant mice were crossed with protamine-cre transgenic mice(24)toremovethefloxedexonand  -actinFlptransgenicmice(25)to remove the neomycin cassette. Genotyping was performed byPCR using the following oligonucleotide primers: SC02105, 5  - AGGACACCACTGGCCCCGAGTCC-3  ; SC02106, 5  -GACG-CACGTCCGCACGTCCACCC-3  ; and SC01114, 5  -TGCCCCT-GAAATGCTGCGCC-3  ,whichgenerateda320-bpWT,a360-bpfloxed,anda300-bpcre-excisedproduct,respectively.Weobtained    -synuclein double-KO mice by crossing the   -synuclein KOmice with previously generated (26)   -synuclein KO mice. Protein Quantitations.  Brain homogenates from four adult litter-mate     /      /   and     /      /   mice (  2 months old) wereanalyzed by quantitative immunoblotting using  125 I-labeled sec-ondary antibodies and PhosphorImager detection (MolecularDynamics) (26) with GDP-dissociation inhibitor (GDI) and vasolin-containing protein (VCP) as internal standards. Electrophysiology.  Vibratome-cut transverse hippocampal slices(400   m thick) from 3- to 5-week-old mice were used for fieldrecordings by using standard procedures (see  Supporting Mate- Abbreviations: GABA,    -aminobutyric acid; KO, knockout; TH, tyrosine hydroxylase. †† To whom correspondence should be addressed. E-mail: thomas.sudhof@utsouthwestern.edu.© 2004 by The National Academy of Sciences of the USA 14966–14971    PNAS    October 12, 2004    vol. 101    no. 41 www.pnas.org  cgi  doi  10.1073  pnas.0406283101   rials and Methods , which is published as supporting informationon the PNAS web site, for a detailed description). OpticalImaging. Dissociatedcorticalneuronsfrom2-to3-day-oldmice were cultured at high density and examined by FM1-43staining at 14 days  in vitro  (27) (see  Supporting Materials and Methods ). Light and Electron Microscopy.  Brain sections from 2-month-oldmice were examined by light and electron microscopy (28). Forquantitation of dopaminergic neurons, coronal brain sections(30   m thick) were stained with an antibody to tyrosine hydrox- ylase (TH; 1:2,000 dilution; Protos Biotech, New York), and theunbiased stereological optical fractionator method was used tocount substantia nigra TH-positive neurons [  n  177–398 neu-rons per animal, with  n    4 male mice per genotype (8 total)(29)]. For quantitation of synaptic parameters, randomizeddigital electron micrographs taken from cultured neurons at day14  in vitro  (30) were examined. Data shown were obtained fromthree independent cultures that contributed equally to the finalnumbers. Quantification of Striatal Monoamines.  The dorsal striatum wasdissected and sonicated in ice-cold 0.1 M perchloric acid con-taining 3,4-dihydroxybenzylamine (Sigma) as an internal stan-dard. After centrifugation, dopamine, serotonin, and metabo-lites were analyzed in the supernatant by HPLC and coulometry(ref. 31; see  Supporting Materials and Methods  for detailedexperimental procedures). Neurotransmitter-Release and  3 H-Dopamine-Uptake Assays.  Synap-tosomes from dorsal striata of WT and mutant mice wereincubated with  3 H-dopamine [20–40 Ci  mmol (1 Ci  37 GBq);PerkinElmer], and uptake was measured as described in ref. 32(see  Supporting Materials and Methods  for detailed experimentalprocedures). Neurotransmitter release was measured with syn-aptosomes loaded with 270 nM  3 H-dopamine (59 Ci  mmol) and8   M [ 14 C]-   -aminobutyric acid (GABA) (240 mCi  mmol).Stimulus-dependent release of dopamine and GABA was mon-itoredinasuperfusionchamber(33,34).Foreachtimepoint,thefractional-releaserateofdopamineandGABAwascalculatedasthe fraction of released radioactivity divided by the amount of radioactivity remaining on the filter. Results Generation of   -Synuclein Knockout (KO) Mice.  By using genomicclones containing the entire murine   -synuclein gene, we con-structed a   -synuclein targeting vector in which the first codingexon is flanked by loxP sites (Fig. 1). We used this targeting vector for homologous recombination experiments with R1embryonic stem cells (26), and we generated mice that trans-mitted the mutant   -synuclein allele through the germ line. Webred the mutant mice with each other and with transgenic micethat express either flp or cre recombinase in the germ line (24,25). In this manner, we generated homozygous mutant mice thatcontained either the initially targeted gene, the targeted gene without the neomycin cassette, or the targeted gene lacking thefirst coding exon (Figs. 1 and 7, which is published as supportinginformation on the PNAS web site).Homozygous mice containing the initially targeted mutant  -synuclein gene (BSC) did not express   -synuclein, as deter-mined by immunoblotting (Fig. 7  B ), probably because the neo-mycin-resistance cassette is present in the mutant gene. Removalof the neomycin-resistance gene cassette with flp recombinaserestored  -synuclein expression, whereas subsequent deletion of  Fig. 2.  Ultrastructural analysis of mutant synapses. (  A ) Representative elec-tron micrographs of synapses in cultured cortical neurons from littermate  -synucleinKO(    /      /   )and    -synucleindouble-KO(    /      /   )mice.(Cal-ibration bar, 100 nm.) ( B ) Frequency distribution of the density of synapticvesicles in synapses from   -synuclein KO (    /      /   ) and     -synuclein dou-ble-KO (    /      /   ) neurons. Synaptic vesicle densities were determined fromrandomized electron micrographs (see  Supporting Materials and Methods ). Fig. 1.  Targeting strategy for the generation of  -synuclein KO mice. Exonsare represented by numbered black boxes, and positions of strategic restric-tion enzyme sites are indicated as follows: E,  Eco RI; B,  Bam HI; X,  Xho I; V, Eco RV; and M,  Mfe I. The locations of the outside probes used for Southernblotting are marked in the WT gene structure, and locations of PCR primersused for genotyping are marked in the targeting-vector structure. The diph-theria toxin (DT) and neomycin-resistance gene cassettes (NEO) that wereintroduced into the targeting vector for negative and positive selection,respectively, are shown as light and dark boxes, respectively. LoxP recombi-nation sites are indicated by gray triangles, and frt recombination sites areindicated by open ovals. Dashed lines indicate insertion of DNA into the WTgene, and dotted lines indicate removal of DNA by flp or cre recombinase. Chandra  et al  . PNAS    October 12, 2004    vol. 101    no. 41    14967       N      E      U      R       O       S       C      I      E      N       C      E  thefirstcodingexonwithcre-recombinaseabolished  -synucleinexpression again. Thus, we produced with our targeting strategyboth conditional   -synuclein KO mice (after flp recombinationonly) and constitutive   -synuclein KO mice (after cre recombi-nation). The conditional KO mice may prove to be useful inanalyses of    -synuclein function in specific brain regions. How-ever, in the present study, we focused on the constitutive  -synuclein KO mice because our goal was to establish thebaseline function of synucleins. Characterization of the     -Synuclein Double-KO Animals.  System-aticbreedingexperimentsrevealedthathomozygous  -synucleinKO mice were viable and fertile, either as single   -synuclein oras    -synuclein double KOs. When we examined the frequencyof genotypes in adult offspring of heterozygous matings, wedetected no decrease in survival of single- or double-mutantmice. [Mendelian ratios of adult surviving mutant mice were asfollows: 1.1:2.0:1.0 for     /      /       /      /       /      /   mice (  n  219), and 1.0:2.0:1.2 for     /      /       /      /       /      /   mice(  n  277).] Because   -synuclein may act as an ‘‘anti-Parkinson’’factor that prevents   -synuclein aggregation and, hence, neuro-degeneration (35), we also tested whether aged   -synuclein KOanimals exhibited signs of neurodegeneration or a shorter life-span. However,   -synuclein KO mice developed no obviousage-dependent phenotype at  1.5 years, were indistinguishablefrom aged WT littermates (data not shown), and had a similarlifespan. These data demonstrate that simultaneous deletion of both synucleins does not impair survival.Next, we used quantitative immunoblotting to measure thelevels of 36 proteins (Table 1, which is published as supportinginformation on the PNAS web site). We compared     -synucleindouble-KOmicewith  -synucleinsingle-KOlittermatecontrol mice because studies have shown that the   -synucleinKO mice exhibit no significant changes in any studied protein(26). The levels of most proteins were not changed significantly, with three exceptions (Table 1): ( i )    -synuclein was increased by  50%in    -synucleindouble-KOmice;( ii )14-3-3  proteinwasincreased by   30%, whereas 14-3-3    protein was decreased by  30%; and ( iii ) complexin was increased by   30%. The   -synuclein increase in the     -synuclein KO probably reflectsa compensatory change, but because    -synuclein levels in theCNS are very low (21), a 50% increase is very small. The 14-3-3proteins are signaling molecules that recognize and dimerize alarge number of target phosphoproteins (36), and they mayinteract with   -synuclein (37). Thus, the changes in 14-3-3proteins in the synuclein KO mice could reflect a functionalrelationship. Complexins are small soluble presynaptic proteinslike synucleins (38). Complexins bind to assembled SNAREcomplexes and regulate neurotransmitter release (39). Theincrease in complexin levels in the synuclein double-KO micemay reflect a functional relationship of synucleins with com-plexins or a compensatory mechanism to make up for the loss of an abundant soluble presynaptic protein in the synuclein KOmice. Synapse Structure in     -Synuclein Double-KO Mice.  We found nostructural abnormalities in the overall brain morphology of     -synuclein double-KO mice by Nissl staining (Fig. 8  A , whichis published as supporting information on the PNAS web site).Electron microscopy of brain sections also failed to revealobvious abnormalities (data not shown). Similarly, in culturedcortical neurons from synuclein KO and control mice, wedetected no deficits in the staining of synaptic markers, such assynaptophysin,rab3a,andsynapsin(Fig.8  B , SupportingMaterials and Methods , and data not shown). We then analyzed thestructure of synapses in cultured neurons from   -synucleinsingle-KO and     -synuclein double-KO mice by quantitativeelectron microscopy (Fig. 2). We detected no significant changesin the presynaptic bouton area (    /      /    0.497  0.37   m 2 ;    /      /     0.398    0.27   m 2 ;  n    114 and 157 synapses,respectively), density of synaptic vesicles (    /      /    135  62synaptic vesicles per   m 2 bouton area;     /      /     149    63synaptic vesicles per  m 2 bouton area;  n  114 and 157 synapses,respectively), active zone length (    /      /     0.37    0.14   m;    /      /     0.40    0.16   m;  n    65 and 95 synapses, respec-tively), or number of docked vesicles (    /      /     11.3    4.9synapses per  m active zone;    /      /    11.4  5.0 synapses per  mactivezone;  n  65and95synapses,respectively).Toexcludethe possibility that deletion of synucleins selectively depletessynaptic vesicles in a subgroup of synapses, we also measured thedistribution of synaptic vesicle densities among different syn-apses, and again we found no significant difference between  -synuclein single-KO synapses and     -synuclein double-KOsynapses (Fig. 2  B ). Synaptic Vesicle Pool Size.  Recent reports (13, 14) have suggestedthat loss of   -synuclein severely decreases the number of reserve vesicles that are mobilized during repetitive stimulation, al-though an earlier study did not detect such a change (12). Todetermine whether synucleins are essential for reserve vesicles, we measured the total size of the synaptic vesicle recycling pool(whichiscomposedofthereadilyreleasablepoolandthereservepool)inneuronsfrom  -and  -synucleinsingle-KO,double-KO,and littermate control mice. By using high-K   stimulation (47mM K   for 90 s), we loaded recycling vesicles in culturedneurons with the styryl dye FM1-43. High-K   stimulation in-duces synaptic vesicle exocytosis and endocytosis, which lead tothe internalization of FM1-43 into synaptic vesicles (40). After washout of free dye, we monitored the release of FM1-43 from Fig. 3.  Functional synaptic vesicle pools in synuclein-deficient synapses.Synapses in cultured cortical neurons were loaded with FM1-43, and the sizeof the pool of actively recycling vesicles was determined as the amount ofFM1-43 fluorescence that could be released upon repeated depolarization(3  90 mM K  for 90 s) in the absence of exogenous FM1-43. Shown is thefrequencydistributionofFM1-43labelingofsynapticboutonsinculturesfrom  -synuclein ( Top ),   -synuclein ( Middle ), and     -synuclein double-KO ( Bot-tom ) neurons (in each case, compared with the corresponding distributionobtained with littermate control mice). 14968    www.pnas.org  cgi  doi  10.1073  pnas.0406283101 Chandra  et al  .  the vesicles when exocytosis is induced by high-frequency fieldstimulation (Fig. 9, which is published as supporting informationon the PNAS web site), and we determined the total amount of FM1-43 fluorescence that is reversibly taken up into synaptic vesicles per bouton, a parameter that reflects the size of the totalrecycling vesicle pool (Fig. 3). We detected no significant changeinthereleaserateofFM1-43orinthesizeoftherecyclingvesiclepool in synuclein-deficient synapses. Thus, consistent with thenormalvesiclenumbersobservedbyelectronmicroscopyandthenormal levels of most synaptic proteins, synuclein single- anddouble-KO mice exhibited no decrease in the size of synaptic vesicle pools. Synaptic Physiology.  The vertebrate-specific expression of synucleins suggests a possible role for synucleins in synapticplasticity (15). To investigate this possibility, we examined fiveprincipal forms of synaptic plasticity in hippocampal slices fromlittermate mice in three genotype comparisons (WT vs.  -synuclein KO mice, WT vs.   -synuclein KO mice, and  -synuclein KO vs.     -synuclein double-KO mice; Figs. 4 and10, which is published as supporting information on the PNAS web site).We first studied two forms of short-term plasticity, paired-pulse facilitation, and posttetanic potentiation by monitoringextracellular synaptic potentials. Paired-pulse facilitation is theenhancement of neurotransmitter release in the second of twoclosely spaced stimuli. Posttetanic potentiation is a longer-lasting enhancement of release induced by a short intense burstof action potentials. For all tested genotype combinations, bothforms of plasticity were unchanged (Fig. 4  A  and  B ).We next probed use-dependent synaptic depression. We ap-plied 14-Hz stimulus trains to WT and mutant hippocampalslices (Fig. 4 C ). In all genotypes, we observed a typical biphasicdecrease in amplitudes during the 14-Hz stimulus train, withoutsignificant differences between WT and KO slices. This result isconsistent with the FM labeling results that also failed to detecta change in the size of recycling and reserve vesicle pools in thesynuclein KO mice. However, it is possible that, in synuclein KOmice, an increase in vesicle recycling compensates for a partialdecrease in pool size. To rule out this possibility, we measuredthe time course with which synaptic responses recover afteruse-dependent depression was induced by high-frequency stim-ulation. Again, we detected no difference between the variousgenotypes(Fig.4  D ),effectivelyrulingoutamajorprobleminthe vesicle numbers, pools, or dynamics in the     -synuclein dou-ble-KO mice. Last, we investigated whether deletion of synucle-ins had an effect on long-term potentiation, but as before, wedetected no difference (Fig. 10). Dopaminergic System.  In Parkinson’s disease, mutations in the  -synuclein gene lead to a selective loss of dopaminergic neuronseven though   -synuclein is widely expressed in the brain. There-fore, we examined whether deletion of    - and  or   -synucleinmay have an effect on the dopaminergic system. We firstquantified striatal levels of dopamine and its metabolites byHPLC. Compared with WT controls, dopamine levels in thestriatum of synuclein double-KO mice, but not of individual   -or   -synuclein KO mice, were decreased by 18% (Fig. 5  A ). Incontrast, the levels of the dopamine metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid Fig. 4.  Hippocampal synaptic plasticity in   - and   -synuclein single- and double-KO mice. All data are taken from recordings at excitatory Schaffercollateral  CA1 pyramidal cell synapses. (  A ) Paired-pulse facilitation. Data show the ratio of the second to the first synaptic response (paired-pulse ratio) to twoclosely spaced stimuli as a function of the interstimulus interval (data are given as number of mice  number of slices;  n  3:9 and 3:8 for    /      /   and WT mice,respectively;  n  1:3 and 2:6 for     /      /   and WT mice, respectively; and  n  2:5 and 2:6 for     /      /   and     /      /   control mice, respectively). ( B ) Posttetanicpotentiation. Potentiation was induced by 30 stimuli at 100 Hz (arrow) in 40   M AP5 (data are given as number of mice  number of slices;  n  3:9 and 3:8 for    /      /   and WT mice, respectively;  n  1:3 and 2:6 for     /      /   and WT mice, respectively; and  n  2:5 and 2:7 for     /      /   and     /      /   mice, respectively).( C  ) Synaptic depression. Synaptic responses during a 14-Hz stimulus train were normalized to the first response (data are given as number of mice  number ofslices; n  3:8and3:8for    /      /   andWTmice,respectively; n  1:3and2:5for    /      /   andWTmice,respectively;and n  2:5and2:6for    /      /   and    /      /   mice, respectively). ( D ) Recovery after synaptic depression induced by a 100-Hz stimulus in the presence of AP5 and 5 mM Ca 2  (data are given as number ofmice  number of slices;  n  3:12 and 3:12 for     /      /   and WT mice, respectively;  n  1:4 and 2:8 for     /      /   and WT mice, respectively; and  n  2:8 and 2:8for    /      /   and    /      /   mice,respectively).Inallgraphs,opensymbolsareplacedabovefilledsymbols,andfilledsymbolsareinvisibleiftheypreciselycoincidewith open symbols. All data are given as mean  SEM. Chandra  et al  . PNAS    October 12, 2004    vol. 101    no. 41    14969       N      E      U      R       O       S       C      I      E      N       C      E  (HVA) were unchanged, suggesting that dopamine catabolism isnot altered dramatically (Fig. 5  B  and data not shown). Inaddition, levels of the monoamine 5-hydroxytryptamine (5-HT) were also unaffected (Fig. 5 C ), indicating that the change indopamine levels is selective. A loss of dopaminergic neurons in the substantia nigra, astypically observed in Parkinson’s disease, could cause the de-crease in dopamine in the synuclein KO mice. To test thispossibility, we stained dopaminergic neurons in the substantianigra with an antibody to TH, and we counted dopaminergicneurons by using stereological methods (Fig. 11, which is pub-lished as supporting information on the PNAS web site). Wefound that the number of TH-positive cells was similar for WTand     -synuclein double-KO male mice (5,428    881 vs.5,418    554 cells per substantia nigra;  n    4 per genotype),suggesting that synucleins are not essential for the developmentor survival of dopaminergic neurons.We next investigated whether the protein levels of key mono-amine biosynthetic enzymes, such as TH, were altered insynuclein KO mice, but we found no significant change (Table 1and Fig. 7). In a final set of experiments, we evaluated the uptakeand the release of dopamine from striatal synaptosomes. Wefound that total dopamine uptake into synaptosomes was onlyslightly decreased in     -synuclein double-KO samples com-pared with WT controls (94    2.9% of controls). For releaseexperiments, we loaded synaptosomes from littermate  -synuclein single-KO mice and     -synuclein double-KO mice with both  3 H-dopamine and [ 14 C]-GABA, and we used a super-fusion system to stimulate neurotransmitter release by successivebrief applications of a high-K   and a hypertonic sucrose solution(33). The fractional-release rate for dopamine and GABA determined in this manner exhibited no difference between  -synuclein single-KO mice and     -synuclein double-KO mice(Fig. 6). Similar results were obtained when double KOs werecompared with WT animals (data not shown). Collectively, thesedata suggest that the synthesis, release, and reuptake of dopa-mine are not impaired dramatically in the synuclein KO animals. Discussion Much progress has been made in elucidating the role of   -synuclein in the pathogenesis of Parkinson’s disease, but thenormal function of this abundant protein remains a mystery. Thehigh concentration of synucleins in presynaptic terminals sug-gested an essential role for synucleins in synapse formation,neurotransmitter release, or synaptic plasticity. Contrary to thisexpectation,thedatapresentedhereshowthatsynucleinsarenotessential for any of these processes. We found that the deletionof    - and   -synucleins did not impair synaptic parameters, suchas the structure of synapse, release of neurotransmitters, mobi-lization of synaptic vesicles, or forms of short- and long-termsynaptic plasticity. Among other findings, these data demon-strate that the lack of a major phenotype in the observed  -synuclein KO mice (12, 26) is not due to redundancy between  - and   -synucleins. However, our results differ from those of Cabin  et al.  (14) who observed a dramatic loss of reserve vesiclesand an increase in synaptic depression in   -synuclein KO mice. A difference in the mouse models that were used may explainthis discrepancy. The   -synuclein KO mice that we (26) and Abeliovich  et al.  (12) generated were made by deleting exons 1and 2, whereas Cabin  et al.  (14) targeted exons 4 and 5 of the  -synuclein gene and may have inadvertently disrupted anunknown downstream gene.Based on  in vitro  studies, it was proposed that   - and  -synuclein have opposite effects on synuclein solubility and Fig. 5.  Levels of dopamine (  A ), 3,4-dihydroxyphenylacetic acid (DOPAC) ( B ),and 5-hydroxytryptamine (5-HT) ( C  ) in the dorsal striatum from synuclein-deficient mice. Monoamine levels were quantified by HPLC ( n  13     /      /   ,13    /      /   ,14    /      /   ,and12WTbrainsfrom2-to4-month-oldmice).DataarenormalizedtoWTlevels(100%)andpresentedasmean  SEM. * , P   0.05,compared with WT, as determined by Student’s  t   test. Fig. 6.  Analysis of dopamine release. Synaptosomes were loaded with 3 H-dopamine (  A ) and  14 C-GABA ( B ) and superfused with normal Krebs bicar-bonate buffer. Release was triggered sequentially by membrane depolariza-tion (30-s pulse of 25 mM K  ) and hypertonic sucrose (30-s pulse of 0.5 Msucrose). Released transmitters were monitored continuously in the superfu-sate. Data are given as mean    SEM from a representative experimentperformed in duplicate and repeated independently three times. 14970    www.pnas.org  cgi  doi  10.1073  pnas.0406283101 Chandra  et al  .
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