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. 2010 Nov 9;107(45):19573-8.
doi: 10.1073/pnas.1005005107. Epub 2010 Oct 25.

αβγ-Synuclein triple knockout mice reveal age-dependent neuronal dysfunction

Becket Greten-Harrison  1 , Manuela Polydoro, Megumi Morimoto-Tomita, Ling Diao, Andrew M Williams, Esther H Nie, Sachin Makani, Ning Tian, Pablo E Castillo, Vladimir L Buchman, Sreeganga S Chandra
Affiliations

αβγ-Synuclein triple knockout mice reveal age-dependent neuronal dysfunction

Becket Greten-Harrison et al. Proc Natl Acad Sci U S A. .

Abstract

Synucleins are a vertebrate-specific family of abundant neuronal proteins. They comprise three closely related members, α-, β-, and γ-synuclein. α-Synuclein has been the focus of intense attention since mutations in it were identified as a cause for familial Parkinson's disease. Despite their disease relevance, the normal physiological function of synucleins has remained elusive. To address this, we generated and characterized αβγ-synuclein knockout mice, which lack all members of this protein family. Deletion of synucleins causes alterations in synaptic structure and transmission, age-dependent neuronal dysfunction, as well as diminished survival. Abrogation of synuclein expression decreased excitatory synapse size by ∼30% both in vivo and in vitro, revealing that synucleins are important determinants of presynaptic terminal size. Young synuclein null mice show improved basic transmission, whereas older mice show a pronounced decrement. The late onset phenotypes in synuclein null mice were not due to a loss of synapses or neurons but rather reflect specific changes in synaptic protein composition and axonal structure. Our results demonstrate that synucleins contribute importantly to the long-term operation of the nervous system and that alterations in their physiological function could contribute to the development of Parkinson's disease.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Generation of synuclein null mice. (A) Western blots of spinal homogenates from wild-type, α-, αβ- and αβγ-synuclein KO mice using isoform-specific antibodies. Valosin-containing protein (VCP) is a loading control. Samples are loaded in duplicate. (B) Immunostaining of cultured wild-type and synuclein null hippocampal neurons for α-synuclein (red) and the dendrite marker MAP2 (green). (C) Body weight of wild-type (αβγ-Syn+/+ blue bar) and synuclein null (αβγ-Syn−/− green bar) mice at 8 wk of age. (D) Survival curves for wild-type (blue triangle) and synuclein null mice (green square). ***P < 0.001.
Fig. 2.
Fig. 2.
Morphometric analysis of synuclein null synapses. (A) Synapse density in CA3 stratum lucidum region of 3-mo-old wild-type αβγ+/+ (blue bar) and synuclein null αβγ−/− mice (green bar) as determined by immunohistochemistry (IHC) with the presynaptic marker synapsin. (B) Presynaptic bouton area of CA3 synapses in wild-type αβγ+/+ and synuclein nulls αβγ−/−. (C) Presynaptic bouton area of CA3 synapses of synuclein nulls αβγ−/− and rescued littermates expressing human wild-type α-synuclein [αβγ−/−; human transgene (htg), red bar]. (D) Presynaptic area of synapses in dissociated hippocampal cultures from wild-type αβγ+/+ and synuclein nulls αβγ−/− mice. (E–I) Analysis of excitatory synapses by electron microscopy (EM). (E) Representative electron micrographs of CA1 synapses from αβγ+/+ and αβγ−/− brains (Scale bar, 250 nm.) (F) Excitatory synapse density of CA3 synapses in wild-type and synuclein null brains. (G) Ultrastructural analysis of CA3 synapses of 3-mo-old wild-type and synuclein null mice. (H) Synapse density in CA1 stratum radiatum of wild-type and synuclein null brains. (I) Ultrastructural analysis of CA1 synapses of wild-type and synuclein null mice. Student's t test. **P < 0.001, ***P < 0.0001.
Fig. 3.
Fig. 3.
Hippocampal physiology of young and aged synuclein null mice. (A and E) I/O curves of Schaffer collateral synapses in 3 mo (A, αβγ-Syn+/+, blue triangle; αβγ-Syn−/−, green square) and 12-mo-old mice (E). (B and F) Data in A and E plotted as a function of fiber volley. (C and G) Paired-pulse facilitation in Schaffer collateral synapses in the hippocampus of 3- (C) and 12-mo-old mice (G). (D and H) Action potential conduction velocity of Schaffer collaterals in 3- (D) and 12-mo-old mice (H). (I and J) I/O curves of Schaffer collateral synapses as a function of stimulus intensity (I) and fiber volley (J) in 3-mo-old mice. [Wild-type αβγ-Syn+/+, blue triangle; synuclein null αβγ-Syn−/−, green square; synuclein null rescued by mouse α-synuclein transgene, αβγ-Syn−/−; mtg, red filled diamond; synuclein null rescued by human α-synuclein transgene αβγ-Syn−/−; htg, red unfilled diamond.] Student's t test *P < 0.05, **P < 0.001, ***P < 0.0001.
Fig. 4.
Fig. 4.
Age-dependent retinal dysfunction in synuclein null mice. ERG analysis of retinal function in wild-type αβγ-Syn+/+ (blue triangle) and synuclein null mice αβγ-Syn−/− mice (green square) in (A) 3 mo and (B) 12 mo. From the ERG traces, the amplitude of the a-wave, b-wave, and the oscillatory potential (OP) was measured (Top, Middle, and Bottom, respectively). (C) Visual placement behavioral analysis of young (2–3 mo) and old (12–18 mo) wild-type (αβγ-Syn+/+; blue bar) and synuclein null (αβγ-Syn−/−; green bar) mice. (D) Inverted grid test analysis of vision and strength. Student's t test *P < 0.05, **P < 0.001, ***P < 0.0001.
Fig. 5.
Fig. 5.
Immunoblotting analysis of wild-type and synuclein null brains. Quantitation of synucleins and the indicated proteins in (A) 3-mo- and (B) 12-mo-old wild-type (blue bar) and αβγ-synuclein KO (green bar) brains. Student's t test *P < 0.05, **P < 0.001, ***P < 0.0001.
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