Presynaptic nerve terminals release neurotransmitters repeatedly, often at high frequency, and in relative isolation from neuronal cell bodies. Repeated release requires cycles of SNARE-complex assembly and disassembly, with continuous generation of reactive SNARE-protein intermediates. Although many forms of neurodegeneration initiate presynaptically, only few pathogenic mechanisms are known, and the functions of presynaptic proteins linked to neurodegeneration, such as α-synuclein, remain unclear. Here, we find that maintenance of continuous presynaptic SNARE-complex assembly required a non-classical chaperone activity mediated by synucleins. Specifically, α-synuclein directly bound to the SNARE-protein synaptobrevin-2/VAMP2, and promoted SNARE-complex assembly. Moreover, triple knockout mice lacking synucleins developed age-dependent neurological impairments, exhibited decreased SNARE-complex assembly, and perished prematurely. Thus, synucleins may function to sustain normal SNAREcomplex assembly in a presynaptic terminal during aging.In presynaptic terminals, neurotransmitter release requires a tightly coordinated membrane fusion machinery whose central components are soluble NSF attachment protein receptor (SNARE) and Sec1/Munc18-like proteins (1-3). Terminals release neurotransmitters thousands of times per minute; during each release reaction, SNARE-complex assembly and disassembly generates highly reactive unfolded SNARE protein intermediates, rendering the terminals potentially vulnerable to activity-dependent degeneration. Indeed, much evidence points to presynaptic terminals as an initiation site for neurodegeneration (4-6), and knockout (KO) of at least one presynaptic chaperone protein, cysteine string protein-α (CSPα), causes fulminant neurodegeneration in mice (7). Synucleins are abundant presynaptic proteins that are expressed from three genes (α-, β-and γ-synuclein; 8). α-Synuclein is involved in neurodegeneration (9-11), and γ-synuclein may contribute to progression of many types of cancer (12). Synucleins may modify neurotransmitter release (13,14), but their physiological functions remain unknown. Strikingly, transgenic expression of α-synuclein abolishes the lethal neurodegeneration induced by KO of CSPα, whereas deletion of endogenous synucleins accelerates this neurodegeneration (15). CSPα KO mice exhibit decreased levels of the SNARE protein SNAP-25 and impaired SNARE-complex assembly, suggesting a link between SNARE-complex assembly and neurodegeneration. α-* To whom correspondence should be addressed: tcs1@stanford.edu. HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptSynuclein rescues SNARE-complex assembly but not SNAP-25 levels (15). This result indicates that α-synuclein may enhance SNARE-protein function, and thereby compensate for the CSPα deletion. To address this hypothesis, we here examine the role of α-synuclein in SNARE-complex assembly and in the maintenance of continuous SNARE-cycling in presynaptic terminals over the lifetime of an animal.We imm...

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.neurodegeneration | loss-of-function | Lewy bodies | ultrastructure | retina S ynucleins are a family of vertebrate-specific proteins with three closely related members, α-, β-, and γ-synuclein (1, 2). They are abundant neuronal proteins and are reported to account for 0.1% of total brain protein (3, 4). Synucleins have overlapping expression patterns and are enriched in presynaptic termini (5, 6). α-Synuclein has been the focus of intense attention since the identification of dominant mutations and gene multiplications that link it to familial Parkinson's disease (PD) (7). Recently, strong ties between the α-synuclein gene and sporadic PD have emerged in genomewide association studies, making α-synuclein the most broadly relevant PD gene (8, 9). Additionally, α-synuclein is the major component of Lewy bodies, the pathological hallmark of PD (10). The presynaptic localization and function of synucleins may also have a bearing on PD, as synapses are lost early in disease progression (11).Analysis of α-, β-, and γ-synuclein sequences reveals a shared, highly conserved N-terminal domain (∼80% identical) with a less conserved acidic C terminus (2). The N-terminal domain has seven imperfect repeats of 11 residues with the consensus sequence XKTKEGVXXXX that binds acidic phospholipid surfaces. Upon lipid binding, synucleins undergo a dramatic change to an α-helical conformation (12-14). α-Synuclein adopts either a conformation consisting of two anti-parallel, amphipathic α-helices with an unfolded C terminus (13-15) or a single extended α-helical structure (16, 17). Human β-and γ-synuclein also adopt the two-helix conformation upon folding (18,19). Together, the high sequence homolog...

The growing body of evidence suggests that intermediate products of a-synuclein aggregation cause death of sensitive populations of neurones, particularly dopaminergic neurones, which is a critical event in the development of Parkinson's disease and other synucleinopathies. The role of two other members of the family, b-synuclein and c-synuclein, in neurodegeneration is less understood. We studied the effect of inactivation of c-synuclein gene on mouse midbrain dopaminergic neurones. Reduced number of dopaminergic neurones was found in substantia nigra pars compacta (SNpc) but not in ventral tegmental area (VTA) of early post-natal and adult c-synuclein null mutant mice. Similar reductions were revealed in a-synuclein and double a-synuclein/c-synuclein null mutant animals. However, in none of these mutants did this lead to significant changes of striatal dopamine or dopamine metabolite levels and motor dysfunction. In all three studied types of null mutants, dopaminergic neurones of SNpc were resistant to methyl-phenyl-tetrahydropyridine (MPTP) toxicity. We propose that both synucleins are important for effective survival of SNpc neurones during critical period of development but, in the absence of these proteins, permanent activation of compensatory mechanisms allow many neurones to survive and become resistant to certain toxic insults.

α-Synuclein is central to the Lewy body neuropathology of Parkinson's disease (PD), a devastating neurodegenerative disorder characterized by numerous motor and non-motor manifestations. The cardinal motor symptoms are linked to death of dopaminergic neurons in the nigrostriatal pathway. Here we ask why these neurons are preferentially susceptible to neurodegeneration in PD and how α-synuclein is involved. To address these questions we bring together recent findings from genome-wide association studies, which reveal the involvement of α-synuclein gene variants in sporadic PD, with recent studies highlighting important roles for α-synuclein in synaptic transmission and dopaminergic neuron physiology. These latest advances add to our understanding of PD etiology and provide a central link between the genetic findings and neurodegeneration observed in sporadic PD.

Optic nerve head (ONH) astrocytes have been proposed to play both protective and deleterious roles in glaucoma. We now show that, within the postlaminar ONH myelination transition zone (MTZ), there are astrocytes that normally express Mac-2 (also known as Lgals3 or galectin-3), a gene typically expressed only in phagocytic cells. Surprisingly, even in healthy mice, MTZ and other ONH astrocytes constitutive internalize large axonal evulsions that contain whole organelles. In mouse glaucoma models, MTZ astrocytes further upregulate Mac-2 expression. During glaucomatous degeneration, there are dystrophic processes in the retina and optic nerve, including the MTZ, which contain protease resistant γ-synuclein. The increased Mac-2 expression by MTZ astrocytes during glaucoma likely depends on this γ-synuclein, as mice lacking γ-synuclein fail to up-regulate Mac-2 at the MTZ after elevation of intraocular pressure. These results suggest the possibility that a newly discovered normal degradative pathway for axons might contribute to glaucomatous neurodegeneration.DBA/2J mice | retinal ganglion cell | Sncg G laucoma, a neurodegenerative disorder that kills retinal ganglion cells (RGCs), affects more than 60 million people and is the second leading cause of blindness worldwide (1). In glaucomatous retinas, both astrocytes and Müller glia increase their reactivity (2, 3). Within the orbital portion of the optic nerve, astrocytes increase in number and reactivity (4). Within the optic nerve head (ONH), astrocytes also increase reactivity in glaucoma animal models and in the human disease (5). These ONH astrocytes are of particular interest as they enwrap axons at the location where damage would account for the arcuate vision field loss characteristic of glaucoma.Here, we identify a spatially discrete population of astrocytes within the ONH, those at the myelination transition zone (MTZ), which express the phagocytosis-related gene Mac-2. Surprisingly, astrocytes throughout the ONH including the MTZ phagocytose large axonal evulsions even in unaffected mice. Mac-2 expression is increased in optic nerve astrocytes upon injury and at the MTZ in two mouse models of glaucoma. In glaucomatous mice, there are protease resistant forms of γ-synuclein, including at the MTZ. Further, mice lacking γ-synuclein fail to up-regulate Mac-2 at the MTZ in response to increased intraocular pressure (IOP). These results suggest the possibility that failure to properly clear axon-derived material at the MTZ, including γ-synuclein, may contribute to axon loss in glaucoma.