Monomeric synucleins generate membrane curvature.

Synucleins are a family of presynaptic membrane binding proteins. α-Synuclein, the principal member of this family, is mutated in familial Parkinson disease. To gain insight into the molecular functions of synucleins, we performed an unbiased proteomic screen and identified synaptic protein changes in αßγ-synuclein knock-out brains. We observed increases in the levels of select membrane curvature sensing/generating proteins. One of the most prominent changes was for the N-BAR protein endophilin A1. Here we demonstrate that the levels of synucleins and endophilin A1 are reciprocally regulated and that they are functionally related. We show that all synucleins can robustly generate membrane curvature similar to endophilins. However, only monomeric but not tetrameric α-synuclein can bend membranes. Further, A30P α-synuclein, a Parkinson disease mutant that disrupts protein folding, is also deficient in this activity. This suggests that synucleins generate membrane curvature through the asymmetric insertion of their N-terminal amphipathic helix. Based on our findings, we propose to include synucleins in the class of amphipathic helix-containing proteins that sense and generate membrane curvature. These results advance our understanding of the physiological function of synucleins.

Differential involvement of the gamma-synuclein in cognitive abilities on the model of knockout mice.

BACKGROUND: Gamma-synuclein is a member of the synuclein family of cytoplasmic, predominantly neuron-specific proteins. Despite numerous evidences for the importance of gamma-synuclein in the control of monoamine homeostasis, cytoskeleton reorganization and chaperone activity, its role in the regulation of cognitive behavior still remain unknown. Our previous study revealed that gamma-synuclein knockout mice are characterized by high habituation scores. Since a number of processes including spatial memory of the environment may affect habituation, in the present study we have carried out behavioral evaluation of spatial and working memory in gamma-synuclein knockout mice. RESULTS: Inactivation of gamma-synuclein gene led to the improvement of working memory in mice as revealed by passive and active avoidance tests. At the same time behavioral tests, designed to assess spatial learning and memory (Morris water maze and Object location tests), showed no differences between gamma-synuclein knockouts and wild type mice. CONCLUSIONS: These findings indicate that young mice with targeted inactivation of gamma-synuclein gene have improved working memory, but not spatial learning and memory. Our results suggest that gamma-synuclein is directly involved in the regulation of cognitive functions.

αßγ-Synuclein triple knockout mice reveal age-dependent neuronal dysfunction.

Synucleins are a vertebrate-specific family of abundant neuronal proteins. They comprise three closely related members, α-, ß-, 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 αßγ-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.

Functional alterations to the nigrostriatal system in mice lacking all three members of the synuclein family.

The synucleins (α, ß, and γ) are highly homologous proteins thought to play a role in regulating neurotransmission and are found abundantly in presynaptic terminals. To overcome functional overlap between synuclein proteins and to understand their role in presynaptic signaling from mesostriatal dopaminergic neurons, we produced mice lacking all three members of the synuclein family. The effect on the mesostriatal system was assessed in adult (4- to 14-month-old) animals using a combination of behavioral, biochemical, histological, and electrochemical techniques. Adult triple-synuclein-null (TKO) mice displayed no overt phenotype and no change in the number of midbrain dopaminergic neurons. TKO mice were hyperactive in novel environments and exhibited elevated evoked release of dopamine in the striatum detected with fast-scan cyclic voltammetry. Elevated dopamine release was specific to the dorsal not ventral striatum and was accompanied by a decrease of dopamine tissue content. We confirmed a normal synaptic ultrastructure and a normal abundance of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complexes in the dorsal striatum. Treatment of TKO animals with drugs affecting dopamine metabolism revealed normal rate of synthesis, enhanced turnover, and reduced presynaptic striatal dopamine stores. Our data uniquely reveal the importance of the synuclein proteins in regulating neurotransmitter release from specific populations of midbrain dopamine neurons through mechanisms that differ from those reported in other neurons. The finding that the complete loss of synucleins leads to changes in dopamine handling by presynaptic terminals specifically in those regions preferentially vulnerable in Parkinson's disease may ultimately inform on the selectivity of the disease process.

Increased striatal dopamine release and hyperdopaminergic-like behaviour in mice lacking both alpha-synuclein and gamma-synuclein.

Alpha-synuclein is intimately involved in the pathogenesis of Parkinson's disease, and has been implicated in the regulation of synthesis, release and reuptake of dopamine (DA). However, mice lacking members of the synuclein family have been reported to display no overt behavioural phenotype. This may be a result of compensatory upregulation of other synucleins during development. Here we report on behaviour and DA synapse function of alpha-synuclein null, gamma-synuclein null, and alpha-gamma-synuclein double-null knockout mice. Double-null mice were hyperactive in a novel environment and alternated at a lower rate in a T-maze spontaneous alternation task, a phenotype reminiscent of mice expressing reduced levels of the DA transporter. To investigate a possible hyperdopaminergic phenotype in alpha-gamma-synuclein double-null mice, we used fast-scan cyclic voltammetry at carbon-fibre microelectrodes to assess DA release and reuptake in striatal slices from wild-type, alpha-null, gamma-null and double-null mice in real time. Double-null mice were found to have a twofold increase in the extracellular concentration of DA detected after discrete electrical stimuli in the striatum. By measuring the rate of reuptake of DA and tissue DA content in these animals, we showed that the observed increase in size of striatal DA transients was not attributable to a decrease in reuptake of DA via the DA transporter, and can not be attributed to an increase in tissue DA levels in the striatum. Rather, we propose that loss of both alpha- and gamma-synuclein causes an increase in release probability from dopaminergic synapses.

The Dopaminergic Dysfunction and Altered Working Memory Performance of Aging Mice Lacking Gamma-synuclein Gene.

BACKGROUND: It was previously shown that inactivation of gamma-synuclein which is a small soluble neuronal protein affects psycho-emotional status and cognitive abilities in knock-out mice. OBJECTIVE: Determine the role of gamma-synuclein inactivation on memory performance in aging animals. METHOD: We used the passive avoidance test and acute amphetamine administration in aging gammasynuclein knock-out mice. RESULTS: As a result, we found moderate aging-unlinked deficit of dopaminergic neurotransmitter system of gamma-synuclein knock-out mice. At the same time, the evidence of progressive synaptic vesicle trafficking machinery impairment was obtained. CONCLUSION: Therefore most likely these dysfunctions are associated with a reduction in the highefficient learning performance in tests that require intact working memory.

Lipid classes and fatty acid patterns are altered in the brain of γ-synuclein null mutant mice.

The well-documented link between α-synuclein and the pathology of common human neurodegenerative diseases has increased attention to the synuclein protein family. The involvement of α-synuclein in lipid metabolism in both normal and diseased nervous system has been shown by many research groups. However, the possible involvement of γ-synuclein, a closely-related member of the synuclein family, in these processes has hardly been addressed. In this study, the effect of γ-synuclein deficiency on the lipid composition and fatty acid patterns of individual lipids from two brain regions has been studied using a mouse model. The level of phosphatidylserine (PtdSer) was increased in the midbrain whereas no changes in the relative proportions of membrane polar lipids were observed in the cortex of γ-synuclein-deficient compared to wild-type (WT) mice. In addition, higher levels of docosahexaenoic acid were found in PtdSer and phosphatidylethanolamine (PtdEtn) from the cerebral cortex of γ-synuclein null mutant mice. These findings show that γ-synuclein deficiency leads to alterations in the lipid profile in brain tissues and suggest that this protein, like α-synuclein, might affect neuronal function via modulation of lipid metabolism.