Synucleins (syns) are a family of proteins involved in several human neurodegenerative diseases and tumors. Since the first syn discovery in the brain of the electric ray Torpedo californica, members of the same family have been identified in all vertebrates and comparative studies have indicated that syn proteins are evolutionary conserved. No counterparts of syns were found in invertebrates suggesting that they are vertebrate-specific proteins. Molecular studies showed that the number of syn members varies among vertebrates. Three genes encode for α-, β- and γ-syn in mammals and birds. However, a variable number of syn genes and encoded proteins is expressed or predicted in fish depending on the species. Among biologically verified sequences, four syn genes were identified in fugu, encoding for α, β and two γ (γ1 and γ2) isoforms, whereas only three genes are expressed in zebrafish, which lacks α-syn gene. The list of "non verified" sequences is much longer and is often found in sequence databases. In this review we provide an overview of published papers and known syn sequences in agnathans and fish that are likely to impact future studies in this field. Indeed, fish models may play a key role in elucidating some of the molecular mechanisms involved in physiological and pathological functions of syn proteins.

Endocannabinoids are among the most powerful modulators of synaptic transmission throughout the nervous system, and yet little is understood about the release of endocannabinoids from postsynaptic compartments. Here we report an unexpected finding that endocannabinoid release requires synucleins, key contributors to Parkinson's disease. We show that endocannabinoids are released postsynaptically by a synuclein-dependent and SNARE-dependent mechanism. Specifically, we found that synuclein deletion blocks endocannabinoid-dependent synaptic plasticity; this block is reversed by postsynaptic expression of wild-type but not of mutant α-synuclein. Whole-cell recordings and direct optical monitoring of endocannabinoid signaling suggest that the synuclein deletion specifically blocks endocannabinoid release. Given the presynaptic role of synucleins in regulating vesicle lifecycle, we hypothesize that endocannabinoids are released via a membrane interaction mechanism. Consistent with this hypothesis, postsynaptic expression of tetanus toxin light chain, which cleaves synaptobrevin SNAREs, also blocks endocannabinoid-dependent signaling. The unexpected finding that endocannabinoids are released via a synuclein-dependent mechanism is consistent with a general function of synucleins in membrane trafficking and adds a piece to the longstanding puzzle of how neurons release endocannabinoids to induce synaptic plasticity.

Synucleins (α, β, γ-synuclein) are a family of abundant presynaptic proteins. α-Synuclein is causally linked to the pathogenesis of Parkinson's disease (PD). In an effort to define their physiological and pathological function or functions, we investigated the effects of deleting synucleins and overexpressing α-synuclein PD mutations, in mice, on synapse architecture using electron microscopy (EM) and cryoelectron tomography (cryo-ET). We show that synucleins are regulators of presynapse size and synaptic vesicle (SV) pool organization. Using cryo-ET, we observed that deletion of synucleins increases SV tethering to the active zone but decreases the inter-linking of SVs by short connectors. These ultrastructural changes were correlated with discrete protein phosphorylation changes in αβγ-synuclein-/- neurons. We also determined that α-synuclein PD mutants (PARK1/hA30P and PARK4/hα-syn) primarily affected presynaptic cytomatrix proximal to the active zone, congruent with previous findings that these PD mutations decrease neurotransmission. Collectively, our results suggest that synucleins are important orchestrators of presynaptic terminal topography.

Synaptic re-uptake of dopamine is dependent on the dopamine transporter (DAT), which is regulated by its distribution to the cell surface. DAT trafficking is modulated by the Parkinson's disease-linked protein alpha-synuclein, but the contribution of synuclein family members beta-synuclein and gamma-synuclein to DAT trafficking is not known. Here we use SH-SY5Y cells as a model of DAT trafficking to demonstrate that all three synucleins negatively regulate cell surface distribution of DAT. Under these conditions the synucleins limit export of DAT from the endoplasmic reticulum (ER) by impairment of the ER-Golgi transition, leading to accumulation of DAT in this compartment. This mechanism for regulating DAT export indirectly through effects on ER and Golgi function represents a previously unappreciated role for the extended synuclein family that is likely applicable to trafficking of the many proteins that rely on the secretory pathway.

Alpha-synuclein (aSyn) plays a central role in Parkinson's disease (PD) and has been extensively studied in the brain. This protein is part of the synuclein family, which is also composed of beta-synuclein (bSyn) and gamma-synuclein (gSyn). In addition to its neurotoxic role, synucleins have important functions in the nervous system, modulating synaptic transmission. Synucleins are expressed in the retina, but they have been poorly characterized. However, there is evidence that they are important for visual function and that they can play a role in retinal degeneration. This study aimed to profile synucleins in the retina of naturally aged mice and to correlate their patterns with specific retinal cells. With aging, we observed a decrease in the thickness of specific retinal layers, accompanied by an increase in glial reactivity. Moreover, the aSyn levels decreased, whereas bSyn increased with aging. The colocalization of both proteins was decreased in the inner plexiform layer (IPL) of the aged retina. gSyn presented an age-related decrease at the inner nuclear layer but was not significantly changed in the ganglion cell layer. The synaptic marker synaptophysin was shown to be preferentially colocalized with aSyn in the IPL with aging. At the same time, aSyn was found to exist at the presynaptic endings of bipolar cells and was affected by aging. Overall, this study suggests that physiological aging can be responsible for changes in the retinal tissue, implicating functional alterations that could affect synuclein family function.

Abnormal α-synuclein aggregates are hallmarks of a number of neurodegenerative diseases. Alpha synuclein and β-synucleins are susceptible to post-translational modification as isoaspartate protein damage, which is regulated in vivo by the action of the repair enzyme protein L-isoaspartyl O-methyltransferase (PIMT). We aged in vitro native α-synuclein, the α-synuclein familial mutants A30P and A53T that give rise to Parkinsonian phenotypes, and β-synuclein, at physiological pH and temperature for a time course of up to 20 days. Resolution of native α-synuclein and β-synuclein by two dimensional techniques showed the accumulation of a number of post-translationally modified forms of both proteins. The levels of isoaspartate formed over the 20 day time course were quantified by exogenous methylation with PIMT using S-Adenosyl-L-[(3)H-methyl]methionine as a methyl donor, and liquid scintillation counting of liberated (3)H-methanol. All α-synuclein proteins accumulated isoaspartate at ∼1% of molecules/day, ∼20 times faster than for β-synuclein. This disparity between rates of isoaspartate was confirmed by exogenous methylation of synucleins by PIMT, protein resolution by one-dimensional denaturing gel electrophoresis, and visualisation of (3)H-methyl esters by autoradiography. Protein silver staining and autoradiography also revealed that α-synucleins accumulated stable oligomers that were resistant to denaturing conditions, and which also contained isoaspartate. Co-incubation of approximately equimolar β-synuclein with α-synuclein resulted in a significant reduction of isoaspartate formed in all α-synucleins after 20 days of ageing. Co-incubated α- and β-synucleins, or α, or β synucleins alone, were resolved by non-denaturing size exclusion chromatography and all formed oligomers of ∼57.5 kDa; consistent with tetramerization. Direct association of α-synuclein with β-synuclein in column fractions or from in vitro ageing co-incubations was demonstrated by their co-immunoprecipitation. These results provide an insight into the molecular differences between α- and β-synucleins during ageing, and highlight the susceptibility of α-synuclein to protein damage, and the potential protective role of β-synuclein.

The synuclein family includes three neuronal proteins, named α-synuclein, β-synuclein, and γ-synuclein, that have peculiar structural features. α-synuclein is largely known for being a key protein in the pathophysiology of Parkinson's disease (PD) and other synucleinopathies, namely, dementia with Lewy bodies and multisystem atrophy. The role of β-synuclein and γ-synuclein is less well understood in terms of physiological functions and potential contribution to human diseases. α-synuclein has been investigated extensively in both cerebrospinal fluid (CSF) and blood as a potential biomarker for synucleinopathies. Recently, great attention has been also paid to β-synuclein, whose CSF and blood levels seem to reflect synaptic damage and neurodegeneration independent of the presence of synucleinopathy. In this review, we aim to provide an overview on the pathophysiological roles of the synucleins. Because γ-synuclein has been poorly investigated in the field of synucleinopathy and its pathophysiological roles are far from being clear, we focus on the interactions between α-synuclein and β-synuclein in PD. We also discuss the role of α-synuclein and β-synuclein as potential biomarkers to improve the diagnostic characterization of synucleinopathies, thus highlighting their potential application in clinical trials for disease-modifying therapies. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

α-synuclein, β-synuclein, and γ-synuclein are abundantly expressed proteins in the vertebrate nervous system. α-synuclein functions in neurotransmitter release by binding to and clustering synaptic vesicles and chaperoning SNARE-complex assembly. Pathologically, aggregates originating from soluble pools of α-synuclein are deposited into Lewy bodies in Parkinson's disease and related synucleinopathies. The functions of β-synuclein and γ-synuclein in presynaptic terminals remain poorly studied. Using in vitro liposome binding studies, circular dichroism spectroscopy, immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments on isolated synaptic vesicles in combination with subcellular fractionation of brains from synuclein mouse models, we show that β-synuclein and γ-synuclein have a reduced affinity toward synaptic vesicles compared with α-synuclein, and that heteromerization of β-synuclein or γ-synuclein with α-synuclein results in reduced synaptic vesicle binding of α-synuclein in a concentration-dependent manner. Our data suggest that β-synuclein and γ-synuclein are modulators of synaptic vesicle binding of α-synuclein and thereby reduce α-synuclein's physiological activity at the neuronal synapse.

Synucleins are small prone to aggregate proteins associated with several neurodegenerative diseases (NDDs), however their role in traumatic brain injury (TBI) is an emerging area of investigation. Using in vitro scratch injury model and in vivo mouse weight-drop model we have found that the injury causes alterations in the expression and localization of synucleins near the damaged area. Before injury, α-synuclein is diffused in the cytoplasm of neurons and γ-synuclein is both in the cytoplasm and nucleus of oligodendrocytes. After the scratch injury of the mixed neuronal and glial culture, α-synuclein forms punctate structures in the cytoplasm of neurons and γ-synuclein is almost completely localized to the nucleus of the oligodendrocytes. Furthermore, the amount of post-translationally modified Met38-oxidized γ-synuclein is increased 3.8 fold 24 h after the scratch. α- and γ-synuclein containing cells increased in the initially cell free scratch zone up to 24 h after the scratch.Intracellular expression and localization of synucleins are also changed in a mouse model of focal closed head injury, using a standardized weight drop device. γ-Synuclein goes from diffuse to punctate staining in a piriform cortex near the amygdala, which may reflect the first steps in the formation of deposits/inclusions. Surprisingly, oxidized γ-synuclein co-localizes with cofilin-actin rods in the thalamus, which are absent in all other regions of the brain. These structures reach their peak amounts 7 days after injury. The changes in γ-synuclein localization are accompanied by injury-induced alterations in the morphology of both astrocytes and neurons.

Synucleins are involved in multiple steps of the neurotransmitter turnover, but the largely normal synaptic function in young adult animals completely lacking synucleins suggests their roles are dispensable for execution of these processes. Instead, they may be utilized for boosting the efficiency of certain molecular mechanisms in presynaptic terminals, with a deficiency of synuclein proteins sensitizing to or exacerbating synaptic malfunction caused by accumulation of mild alterations, which are commonly associated with aging. Although functional redundancy within the family has been reported, it is unclear whether the remaining synucleins can fully compensate for the deficiency of a lost family member or whether some functions are specific for a particular member. We assessed several structural and functional characteristics of the nigrostriatal system of mice lacking members of the synuclein family in every possible combination and demonstrated that stabilization of the striatal dopamine level depends on the presence of α-synuclein and cannot be compensated by other family members, whereas β-synuclein is required for efficient maintenance of animal's balance and coordination in old age.

Maternal alcohol consumption is one of the strong predictive factors of alcohol use and consequent abuse; however, investigations of sex differences in response to prenatal alcohol exposure (PAE) are limited. Here we compared the effects of PAE throughout gestation on alcohol preference, state anxiety and mRNA expression of presynaptic proteins α-, β- and γ-synucleins in the brain of adult (PND60) male and female Wistar rats. Total RNA was isolated from the hippocampus, midbrain and hypothalamus and mRNA levels were assessed with quantitative RT-PCR. Compared with naïve males, naïve female rats consumed more alcohol in "free choice" paradigm (10% ethanol vs. water). At the same time, PAE produced significant increase in alcohol consumption and preference in males but not in females compared to male and female naïve groups, correspondingly. We found significantly lower α-synuclein mRNA levels in the hippocampus and midbrain of females compared to males and significant decrease in α-synuclein mRNA in these brain areas in PAE males, but not in females compared to the same sex controls. These findings indicate that the impact of PAE on transcriptional regulation of synucleins may be sex-dependent, and in males' disruption in α-synuclein mRNA expression may contribute to increased vulnerability to alcohol-associated behavior.

Synucleins comprise a family of small proteins highly expressed in the nervous system of vertebrates and involved in various intraneuronal processes. The malfunction of alpha-synuclein is one of the key events in pathogenesis of Parkinson disease and certain other neurodegenerative diseases, and there is a growing body of evidence that malfunction of other two synucleins might be involved in pathological processes in the nervous system. The modulation of various presynaptic mechanisms of neurotransmission is an important function of synucleins, and therefore, it is feasible that their deficiency might affect global electrical activity detected of the brain. However, the effects of the loss of synucleins on the frequency spectra of electroencephalograms (EEGs) have not been systematically studied so far. In the current study, we assessed changes in such spectra in single-, double- and triple-knockout mice lacking alpha-, beta- and gamma-synucleins in all possible combinations. EEGs were recorded from the motor cortex, the putamen, the ventral tegmental area and the substantia nigra of 78 3-month-old male mice from seven knockout groups maintained on the C57BL/6J genetic background, and 10 wild-type C57BL/6J mice for 30 min before and for 60 min after the systemic injection of a DA receptor agonist, apomorphine (APO). We found that almost any variant of synuclein deficiency causes multiple changes in both basal and APO-induced EEG oscillation profiles. Therefore, it is not the absence of any particular synuclein but rather a disbalance of synucleins that causes widespread changes in EEG spectral profiles.

Glaucoma, one of the leading causes of irreversible blindness worldwide, is a group of disorders characterized by progressive retinal ganglion cell (RGC) loss. Synucleins, a family of small proteins, have been of interest in studies of neurodegeneration and CNS. However, their roles and functions in glaucoma are still not completely understood and remain to be explored. Our previous studies showed that α-synuclein and H2S play a pivotal role in glaucoma. This study aims to (1) elucidate the potential roles and functions of synucleins in glaucoma throughout aging, (2) investigate the interaction between the synucleins and H2S, and better understand the mechanism of H2S in neuroprotection.

α-Synuclein is expressed at high levels at presynaptic terminals, but defining its role in the regulation of neurotransmission under physiologically relevant conditions has proven elusive. We report that, in vivo, α-synuclein is responsible for the facilitation of dopamine release triggered by action potential bursts separated by short intervals (seconds) and a depression of release with longer intervals between bursts (minutes). These forms of presynaptic plasticity appear to be independent of the presence of β- and γ-synucleins or effects on presynaptic calcium and are consistent with a role for synucleins in the enhancement of synaptic vesicle fusion and turnover. These results indicate that the presynaptic effects of α-synuclein depend on specific patterns of neuronal activity.

In neuronal synapses, neurotransmitter-loaded vesicles fuse with presynaptic plasma membrane in a complex sequence of tightly regulated events. The assembly of specialized SNARE complexes plays a pivotal role in this process. The function of the chaperone cysteine string protein α (CSPα) is important for synaptic SNARE complex formation, and mice lacking this protein develop severe synaptic dysfunction and neurodegeneration that lead to their death within 3 months after birth. Another presynaptic protein, α-synuclein, also potentiates SNARE complex formation, and its overexpression rescues the phenotype of CSPα null mutant mice, although these two proteins use different mechanisms to achieve this effect. α-Synuclein is a member of a family of three related proteins whose structural similarity suggests functional redundancy. Here, we assessed whether γ-synuclein shares the ability of α-synuclein to bind synaptic vesicles and ameliorate neurodegeneration caused by CSPα deficiency in vivo. Although the N-terminal lipid-binding domains of the two synucleins showed similar affinity for purified synaptic vesicles, the C-terminal domain of γ-synuclein was not able to interact with synaptobrevin-2/VAMP2. Consequently, overexpression of γ-synuclein did not have any noticeable effect on the phenotype of CSPα null mutant mice. Our data suggest that the functions of α- and γ-synucleins in presynaptic terminals are not fully redundant.

Several neurodegenerative diseases are classified as proteopathies as they are associated with the aggregation of misfolded proteins. Synucleinopathies are a group of neurodegenerative disorders associated with abnormal deposition of synucleins. α-Synucleinopathies include Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Recently accumulation of another member of the synuclein family- γ-synuclein in neurodegenerative diseases compelled the introduction of the term γ-synucleinopathy. The formation of aggregates and deposits of γ-synuclein is facilitated after its oxidation at methionine 38 (Met38).

Mechanistic understanding of nucleation dependent polymerization by α-synuclein (α-Syn) into toxic oligomers and amyloids is important for the drug development against Parkinson's disease. However the structural and morphological characterization during nucleation and subsequent fibrillation process of α-Syn is not clearly understood. Using a variety of complementary biophysical techniques monitoring entire pathway of nine different synucleins, we found that transition of unstructured conformation into β-sheet rich fibril formation involves helix-rich intermediates. These intermediates are common for all aggregating synucleins, contain high solvent-exposed hydrophobic surfaces, are cytotoxic to SHSY-5Y cells and accelerate α-Syn aggregation efficiently. A multidimensional NMR study characterizing the intermediate accompanied with site-specific fluorescence study suggests that the N-terminal and central portions mainly participate in the helix-rich intermediate formation while the C-terminus remained in an extended conformation. However, significant conformational transitions occur at the middle and at the C-terminus during helix to β-sheet transition as evident from Trp fluorescence study. Since partial helix-rich intermediates were also observed for other amyloidogenic proteins such as Aβ and IAPP, we hypothesize that this class of intermediates may be one of the important intermediates for amyloid formation pathway by many natively unstructured protein/peptides and represent a potential target for drug development against amyloid diseases.

Despite numerous evidences for neurotoxicity of overexpressed alpha-synuclein, a protective function was suggested for endogenous alpha-synuclein and other members of the synuclein family. This protective role is most important for and evident in presynaptic terminals, where synucleins are normally accumulated. However, mice lacking synucleins display no adverse phenotype. In particular, no significant changes in striatal dopamine metabolism and only subtle deficit of dopaminergic neurons in the substantia nigra were found in juvenile or adult mice. To assess whether aging and synuclein deficiency may have additive detrimental effect on the nigrostriatal system, we studied dopaminergic neurons of the substantia nigra and their striatal synapses in 24-26-month-old alpha-synuclein and gamma-synuclein null mutant mice. Significant approximately 36% reduction of the striatal dopamine was found in aging alpha-synuclein, but not gamma-synuclein null mutant mice when compared to age-matching wild type mice. This was accompanied by the reduction of TH-positive fibers in the striatum and decrease of striatal levels of TH and DAT. However, no progressive loss of TH-positive neurons was revealed in the substantia nigra of synuclein-deficient aging animals. Our results are consistent with a hypothesis that alpha-synuclein is important for normal function and integrity of synapses, and suggest that in the aging nervous system dysfunction of this protein could become a predisposition factor for the development of nigrostriatal pathology.

Focal cortical dysplasia (FCD) IIb and tuberous sclerosis complex (TSC) are common causes of drug-resistant epilepsy in children. However, the etiologies related to the development of FCD IIb and TSC are not fully understood. α-synuclein (α-syn) is a member of synucleins family that plays crucial roles in modulating synaptic transmission in central nervous system. Here, we explored the expression profiles and potential pathogenic functions of α-syn in cortical lesions of epileptic patients with FCD IIb and TSC.

The cytosolic proteins synucleins and synapsins are thought to play cooperative roles in regulating synaptic vesicle (SV) recycling, but mechanistic insight is lacking. Here we identify the synapsin E-domain as an essential functional binding-partner of α-synuclein (α-syn). Synapsin E-domain allows α-syn functionality, binds to α-syn, and is necessary and sufficient for enabling effects of α-syn at the synapse. Together with previous studies implicating the E-domain in clustering SVs, our experiments advocate a cooperative role for these two proteins in maintaining physiologic SV clusters.