β-synuclein potentiates synaptic vesicle dopamine uptake and rescues dopaminergic neurons from MPTP-induced death in the absence of other synucleins

Synucleins, a family of three proteins highly expressed in neurons, are predominantly known for the direct involvement of α-synuclein in the etiology and pathogenesis of Parkinson''s and certain other neurodegenerative diseases, but their precise physiological functions are still not fully understood. Previous studies have demonstrated the importance of α-synuclein as a modulator of various mechanisms implicated in chemical neurotransmission, but information concerning the involvement of other synuclein family members, β-synuclein and γ-synuclein, in molecular processes within presynaptic terminals is limited. Here, we demonstrated that the vesicular monoamine transporter 2–dependent dopamine uptake by synaptic vesicles isolated from the striatum of mice lacking β-synuclein is significantly reduced. Reciprocally, reintroduction, either in vivo or in vitro, of β-synuclein but not α-synuclein or γ-synuclein improves uptake by triple α/β/γ-synuclein–deficient striatal vesicles. We also showed that the resistance of dopaminergic neurons of the substantia nigra pars compacta to subchronic administration of the Parkinson''s disease–inducing prodrug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine depends on the presence of β-synuclein but only when one or both other synucleins are absent. Furthermore, proteomic analysis of synuclein-deficient synaptic vesicles versus those containing only β-synuclein revealed differences in their protein compositions. We suggest that the observed potentiation of dopamine uptake by β-synuclein might be caused by different protein architecture of the synaptic vesicles. It is also feasible that such structural changes improve synaptic vesicle sequestration of 1-methyl-4-phenylpyridinium, a toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which would explain why dopaminergic neurons expressing β-synuclein and lacking α-synuclein and/or γ-synuclein are resistant to this neurotoxin.     

Contrasting Effects of α-Synuclein and γ-Synuclein on the Phenotype of Cysteine String Protein α (CSPα) Null Mutant Mice Suggest Distinct Function of these Proteins in Neuronal Synapses

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.     

N-terminal Acetylation Stabilizes N-terminal Helicity in Lipid- and Micelle-bound α-Synuclein and Increases Its Affinity for Physiological Membranes

The Parkinson disease protein α-synuclein is N-terminally acetylated, but most in vitro studies have been performed using unacetylated α-synuclein. Binding to lipid membranes is considered key to the still poorly understood function of α-synuclein. We report the effects of N-terminal acetylation on α-synuclein binding to lipid vesicles of different composition and curvature and to micelles composed of the detergents β-octyl-glucoside (BOG) and SDS. In the presence of SDS, N-terminal acetylation results in a slightly increased helicity for the N-terminal ∼10 residues of the protein, likely due to the stabilization of N-terminal fraying through the formation of a helix cap motif. In the presence of BOG, a detergent used in previous isolations of helical oligomeric forms of α-synuclein, the N-terminally acetylated protein adopts a novel conformation in which the N-terminal ∼30 residues bind the detergent micelle in a partly helical conformation, whereas the remainder of the protein remains unbound and disordered. Binding of α-synuclein to lipid vesicles with high negative charge content is essentially unaffected by N-terminal acetylation irrespective of curvature, but binding to vesicles of lower negative charge content is increased, with stronger binding observed for vesicles with higher curvature. Thus, the naturally occurring N-terminally acetylated form of α-synuclein exhibits stabilized helicity at its N terminus and increased affinity for lipid vesicles similar to synaptic vesicles, a binding target of the protein in vivo. Furthermore, the novel BOG-bound state of N-terminally acetylated α-synuclein may serve as a model of partly helical membrane-bound intermediates with a role in α-synuclein function and dysfunction.     

Membrane Curvature Induction and Tubulation Are Common Features of Synucleins and Apolipoproteins

Synucleins and apolipoproteins have been implicated in a number of membrane and lipid trafficking events. Lipid interaction for both types of proteins is mediated by 11 amino acid repeats that form amphipathic helices. This similarity suggests that synucleins and apolipoproteins might have comparable effects on lipid membranes, but this has not been shown directly. Here, we find that α-synuclein, β-synuclein, and apolipoprotein A-1 have the conserved functional ability to induce membrane curvature and to convert large vesicles into highly curved membrane tubules and vesicles. The resulting structures are morphologically similar to those generated by amphiphysin, a curvature-inducing protein involved in endocytosis. Unlike amphiphysin, however, synucleins and apolipoproteins do not require any scaffolding domains and curvature induction is mediated by the membrane insertion and wedging of amphipathic helices alone. Moreover, we frequently observed that α-synuclein caused membrane structures that had the appearance of nascent budding vesicles. The ability to function as a minimal machinery for vesicle budding agrees well with recent findings that α-synuclein plays a role in vesicle trafficking and enhances endocytosis. Induction of membrane curvature must be under strict regulation in vivo; however, as we find it can also cause disruption of membrane integrity. Because the degree of membrane curvature induction depends on the concerted action of multiple proteins, controlling the local protein density of tubulating proteins may be important. How cellular safeguarding mechanisms prevent such potentially toxic events and whether they go awry in disease remains to be determined.     

Metformin lowers Ser-129 phosphorylated α-synuclein levels via mTOR-dependent protein phosphatase 2A activation

Phospho-Ser129 α-synuclein is the modified form of α-synuclein that occurs most frequently within Parkinson''s disease pathological inclusions. Here we demonstrate that the antidiabetic drug metformin significantly reduces levels of phospho-Ser129 α-synuclein and the ratio of phospho-Ser129 α-synuclein to total α-synuclein. This effect was documented in vitro in SH-SY5Y and HeLa cells as well as in primary cultures of hippocampal neurons. In vitro work also elucidated the mechanisms underlying metformin''s action. Following metformin exposure, decreased phospho-Ser129 α-synuclein was not strictly dependent on induction of AMP-activated protein kinase, a primary target of the drug. On the other hand, metformin-induced phospho-Ser129 α-synuclein reduction was consistently associated with inhibition of mammalian target of rapamycin (mTOR) and activation of protein phosphatase 2A (PP2A). Evidence supporting a key role of mTOR/PP2A signaling included the finding that, similar to metformin, the canonical mTOR inhibitor rapamycin was capable of lowering the ratio of phospho-Ser129 α-synuclein to total α-synuclein. Furthermore, no decrease in phosphorylated α-synuclein occurred with either metformin or rapamycin when phosphatase activity was inhibited, supporting a direct relationship between mTOR inhibition, PP2A activation and protein dephosphorylation. A final set of experiments confirmed the effectiveness of metformin in vivo in wild-type C57BL/6 mice. Addition of the drug to food or drinking water lowered levels of phospho-Ser129 α-synuclein in the brain of treated animals. These data reveal a new mechanism leading to α-synuclein dephosphorylation that could be targeted for therapeutic intervention by drugs like metformin and rapamycin.     

Alcohol-Related Brain Damage in Humans

Chronic excessive alcohol intoxications evoke cumulative damage to tissues and organs. We examined prefrontal cortex (Brodmann’s area (BA) 9) from 20 human alcoholics and 20 age, gender, and postmortem delay matched control subjects. H & E staining and light microscopy of prefrontal cortex tissue revealed a reduction in the levels of cytoskeleton surrounding the nuclei of cortical and subcortical neurons, and a disruption of subcortical neuron patterning in alcoholic subjects. BA 9 tissue homogenisation and one dimensional polyacrylamide gel electrophoresis (PAGE) proteomics of cytosolic proteins identified dramatic reductions in the protein levels of spectrin β II, and α- and β-tubulins in alcoholics, and these were validated and quantitated by Western blotting. We detected a significant increase in α-tubulin acetylation in alcoholics, a non-significant increase in isoaspartate protein damage, but a significant increase in protein isoaspartyl methyltransferase protein levels, the enzyme that triggers isoaspartate damage repair in vivo. There was also a significant reduction in proteasome activity in alcoholics. One dimensional PAGE of membrane-enriched fractions detected a reduction in β-spectrin protein levels, and a significant increase in transmembranous α3 (catalytic) subunit of the Na⁺,K⁺-ATPase in alcoholic subjects. However, control subjects retained stable oligomeric forms of α-subunit that were diminished in alcoholics. In alcoholics, significant loss of cytosolic α- and β-tubulins were also seen in caudate nucleus, hippocampus and cerebellum, but to different levels, indicative of brain regional susceptibility to alcohol-related damage. Collectively, these protein changes provide a molecular basis for some of the neuronal and behavioural abnormalities attributed to alcoholics.     

Parkin Deficiency Delays Motor Decline and Disease Manifestation in a Mouse Model of Synucleinopathy

In synucleinopathies, including Parkinson''s disease, partially ubiquitylated α-synuclein species phosphorylated on serine 129 (P^S129-α-synuclein) accumulate abnormally. Parkin, an ubiquitin-protein ligase that is dysfunctional in autosomal recessive parkinsonism, protects against α-synuclein-mediated toxicity in various models.We analyzed the effects of Parkin deficiency in a mouse model of synucleinopathy to explore the possibility that Parkin and α-synuclein act in the same biochemical pathway. Whether or not Parkin was present, these mice developed an age-dependent neurodegenerative disorder preceded by a progressive decline in performance in tasks predictive of sensorimotor dysfunction. The symptoms were accompanied by the deposition of P^S129-α-synuclein but not P^S87-α-synuclein in neuronal cell bodies and neuritic processes throughout the brainstem and the spinal cord; activation of caspase 9 was observed in 5% of the P^S129-α-synuclein-positive neurons. As in Lewy bodies, ubiquitin-immunoreactivity, albeit less abundant, was invariably co-localized with P^S129-α-synuclein. During late disease stages, the disease-specific neuropathological features revealed by ubiquitin- and P^S129-α-synuclein-specific antibodies were similar in mice with or without Parkin. However, the proportion of P^S129-α-synuclein-immunoreactive neuronal cell bodies and neurites co-stained for ubiquitin was lower in the absence than in the presence of Parkin, suggesting less advanced synucleinopathy. Moreover, sensorimotor impairment and manifestation of the neurodegenerative phenotype due to overproduction of human α-synuclein were significantly delayed in Parkin-deficient mice.These findings raise the possibility that effective compensatory mechanisms modulate the phenotypic expression of disease in parkin-related parkinsonism.     

Human β-Synuclein Rendered Fibrillogenic by Designed Mutations

Filamentous inclusions made of α-synuclein are found in nerve cells and glial cells in a number of human neurodegenerative diseases, including Parkinson disease, dementia with Lewy bodies, and multiple system atrophy. The assembly and spreading of these inclusions are likely to play an important role in the etiology of common dementias and movement disorders. Both α-synuclein and the homologous β-synuclein are abundantly expressed in the central nervous system; however, β-synuclein is not present in the pathological inclusions. Previously, we observed a poor correlation between filament formation and the presence of residues 73–83 of α-synuclein, which are absent in β-synuclein. Instead, filament formation correlated with the mean β-sheet propensity, charge, and hydrophilicity of the protein (global physicochemical properties) and β-strand contiguity calculated by a simple algorithm of sliding averages (local physicochemical property). In the present study, we rendered β-synuclein fibrillogenic via one set of point mutations engineered to enhance global properties and a second set engineered to enhance predominantly β-strand contiguity. Our findings show that the intrinsic physicochemical properties of synucleins influence their fibrillogenic propensity via two distinct but overlapping modalities. The implications for filament formation and the pathogenesis of neurodegenerative diseases are discussed.     

5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) Effect on Glucose Production,but Not Energy Metabolism,Is Independent of Hepatic AMPK in Vivo

Metabolic stress, as well as several antidiabetic agents, increases hepatic nucleotide monophosphate (NMP) levels, activates AMP-activated protein kinase (AMPK), and suppresses glucose production. We tested the necessity of hepatic AMPK for the in vivo effects of an acute elevation in NMP on metabolism. 5-Aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR; 8 mg·kg⁻¹·min⁻¹)-euglycemic clamps were performed to elicit an increase in NMP in wild type (α1α2^lox/lox) and liver-specific AMPK knock-out mice (α1α2^lox/lox + Albcre) in the presence of fixed glucose. Glucose kinetics were equivalent in 5-h fasted α1α2^lox/lox and α1α2^lox/lox + Albcre mice. AMPK was not required for AICAR-mediated suppression of glucose production and increased glucose disappearance. These results demonstrate that AMPK is unnecessary for normal 5-h fasting glucose kinetics and AICAR-mediated inhibition of glucose production. Moreover, plasma fatty acids and triglycerides also decreased independently of hepatic AMPK during AICAR administration. Although the glucoregulatory effects of AICAR were shown to be independent of AMPK, these studies provide in vivo support for the AMPK energy sensor paradigm. AICAR reduced hepatic energy charge by ∼20% in α1α2^lox/lox, which was exacerbated by ∼2-fold in α1α2^lox/lox + Albcre. This corresponded to a ∼6-fold rise in AMP/ATP in α1α2^lox/lox + Albcre. Consistent with the effects on adenine nucleotides, maximal mitochondrial respiration was ∼30% lower in α1α2^lox/lox + Albcre than α1α2^lox/lox livers. Mitochondrial oxidative phosphorylation efficiency was reduced by 25%. In summary, these results demonstrate that the NMP capacity to inhibit glucose production in vivo is independent of liver AMPK. In contrast, AMPK promotes mitochondrial function and protects against a more precipitous fall in ATP during AICAR administration.     

Importance of the Voltage Dependence of Cardiac Na/K ATPase Isozymes

Cardiac cells express more than one isoform of the Na, K-ATPase (NKA), the heteromeric enzyme that creates the Na⁺ and K⁺ gradients across the plasmalemma. Cardiac isozymes contain one catalytic α-subunit isoform (α1, α2, or α3) associated with an auxiliary β-subunit isoform (β1 or β2). Past studies using biochemical approaches have revealed minor kinetic differences between isozymes formed by different α-β isoform combinations; these results make it difficult to understand the physiological requirement for multiple isoforms. In intact cells, however, NKA enzymes operate in a more complex environment, which includes a substantial transmembrane potential. We evaluated the voltage dependence of human cardiac NKA isozymes expressed in Xenopus oocytes, and of native NKA isozymes in rat ventricular myocytes, using normal mammalian physiological concentrations of Na⁺_o and K⁺_o. We demonstrate that although α1 and α3 pumps are functional at all physiologically relevant voltages, α2β1 pumps and α2β2 pumps are inhibited by ∼75% and ∼95%, respectively, at resting membrane potentials, and only activate appreciably upon depolarization. Furthermore, phospholemman (FXYD1) inhibits pump function without significantly altering the pump’s voltage dependence. Our observations provide a simple explanation for the physiological relevance of the α2 subunit (∼20% of total α subunits in rat ventricle): they act as a reserve and are recruited into action for extra pumping during the long-lasting cardiac action potential, where most of the Na⁺ entry occurs. This strong voltage dependence of α2 pumps also helps explain how cardiotonic steroids, which block NKA pumps, can be a beneficial treatment for heart failure: by only inhibiting the α2 pumps, they selectively reduce NKA activity during the cardiac action potential, leading to an increase in systolic Ca²⁺, due to reduced extrusion through the Na/Ca exchanger, without affecting resting Na⁺ and Ca²⁺ concentrations.     

Tissue transglutaminase modulates alpha-synuclein oligomerization

We have studied the interaction of the enzyme tissue transglutaminase (tTG), catalyzing cross-link formation between protein-bound glutamine residues and primary amines, with Parkinson's disease-associated α-synuclein protein variants at physiologically relevant concentrations. We have, for the first time, determined binding affinities of tTG for wild-type and mutant α-synucleins using surface plasmon resonance approaches, revealing high-affinity nanomolar equilibrium dissociation constants. Nanomolar tTG concentrations were sufficient for complete inhibition of fibrillization by effective α-synuclein cross-linking, resulting predominantly in intramolecularly cross-linked monomers accompanied by an oligomeric fraction. Since oligomeric species have a pathophysiological relevance we further investigated the properties of the tTG/α-synuclein oligomers. Atomic force microscopy revealed morphologically similar structures for oligomers from all α-synuclein variants; the extent of oligomer formation was found to correlate with tTG concentration. Unlike normal α-synuclein oligomers the resultant structures were extremely stable and resistant to GdnHCl and SDS. In contrast to normal β-sheet-containing oligomers, the tTG/α-synuclein oligomers appear to be unstructured and are unable to disrupt phospholipid vesicles. These data suggest that tTG binds equally effective to wild-type and disease mutant α-synuclein variants. We propose that tTG cross-linking imposes structural constraints on α-synuclein, preventing the assembly of structured oligomers required for disruption of membranes and for progression into fibrils. In general, cross-linking of amyloid forming proteins by tTG may prevent the progression into pathogenic species.     

The AKT modulator A-443654 reduces α-synuclein expression and normalizes ER stress and autophagy

Accumulation of α-synuclein is a main underlying pathological feature of Parkinson’s disease and α-synucleinopathies, for which lowering expression of the α-synuclein gene (SNCA) is a potential therapeutic avenue. Using a cell-based luciferase reporter of SNCA expression we performed a quantitative high-throughput screen of 155,885 compounds and identified A-443654, an inhibitor of the multiple functional kinase AKT, as a potent inhibitor of SNCA. HEK-293 cells with CAG repeat expanded ATXN2 (ATXN2-Q58 cells) have increased levels of α-synuclein. We found that A-443654 normalized levels of both SNCA mRNA and α-synuclein monomers and oligomers in ATXN2-Q58 cells. A-443654 also normalized levels of α-synuclein in fibroblasts and iPSC-derived dopaminergic neurons from a patient carrying a triplication of the SNCA gene. Analysis of autophagy and endoplasmic reticulum stress markers showed that A-443654 successfully prevented α-synuclein toxicity and restored cell function in ATXN2-Q58 cells, normalizing the levels of mTOR, LC3-II, p62, STAU1, BiP, and CHOP. A-443654 also decreased the expression of DCLK1, an inhibitor of α-synuclein lysosomal degradation. Our study identifies A-443654 and AKT inhibition as a potential strategy for reducing SNCA expression and treating Parkinson’s disease pathology.     

Aggregated α-synuclein and complex I deficiency: exploration of their relationship in differentiated neurons

α-Synuclein becomes misfolded and aggregated upon damage by various factors, for example, by reactive oxygen species. These aggregated forms have been proposed to have differential toxicities and their interaction with mitochondria may cause dysfunction within this organelle that contributes to the pathogenesis of Parkinson''s disease (PD). In particular, the association of α-synuclein with mitochondria occurs through interaction with mitochondrial complex I and importantly defects of this protein have been linked to the pathogenesis of PD. Therefore, we investigated the relationship between aggregated α-synuclein and mitochondrial dysfunction, and the consequences of this interaction on cell survival. To do this, we studied the effects of α-synuclein on cybrid cell lines harbouring mutations in either mitochondrial complex I or IV. We found that aggregated α-synuclein inhibited mitochondrial complex I in control and complex IV-deficient cells. However, when aggregated α-synuclein was applied to complex I-deficient cells, there was no additional inhibition of mitochondrial function or increase in cell death. This would suggest that as complex I-deficient cells have already adapted to their mitochondrial defect, the subsequent toxic effects of α-synuclein are reduced.The pathological hallmark of Parkinson''s disease (PD) is the presence of α-synuclein aggregates, particularly within the substantia nigra (SN). These aggregations take the form of intracellular Lewy bodies, and also neuritic aggregations. However, both the effect of these inclusions on neuronal survival and the toxicity of different forms of α-synuclein are still debated. To aggregate α-synuclein must undergo a conformational change, however, the mechanism behind this change and subsequent aggregation in PD remains to be determined.Mutations within the α-synuclein gene (SNCA (MIM 163890)) were the first to be associated with autosomal dominant PD, while more recently genome-wide association studies have suggested that single-nucleotide polymorphisms in this gene are important for sporadic PD. A widely expressed protein α-synuclein is important for synaptic vesicle recycling and the modulation of dopamine transmission within SN neurons.^{1, 2, 3, 4, 5, 6, 7, 8} It interacts with curved cellular membranes including those of mitochondria suggesting a possible mode of its toxicity,^{9, 10, 11} and can be imported into mitochondria in an energy-dependent manner.⁹ The accumulation of α-synuclein within mitochondria leads to complex I impairment, decreased mitochondrial membrane potential (ΔΨ_m) and increased reactive oxygen species (ROS) production. The occurrence of these changes is also dependent on calcium homoeostasis.^{9, 12, 13}Mitochondrial dysfunction has also been heavily implicated in the pathogenesis of PD. Early studies showed a decrease in mitochondrial complex I in the SN of PD patients and studies involving the inhibition of this complex replicate many of the features of this disease. In addition, SN neurons show high levels of mitochondrial DNA deletions in old age,^{14, 15} which lead to respiratory deficiency, and the environment of the SN is believed to be particularly oxidative due to a number of processes, including the metabolism of dopamine. More recently a number of genes known to cause autosomal recessive forms of PD have been shown to encode proteins with functions associated with mitochondrial turnover (Parkin/Pink1 (MIM 602544, MIM 608309)) or oxidative stress (DJ-1 (MIM 602533)). However, the link between these two processes and the loss of dopaminergic neurons in PD remains to be elucidated.Several hypotheses have been suggested for what might cause α-synuclein to undergo the conformational change into more aggregate prone forms, from oxidative stress to gene mutations. Furthermore, the accumulation of mitochondrial DNA (mtDNA) mutations and dysfunctional mitochondria with advancing age are likely to have an effect on oxidative stress levels within the SN, which might contribute further to the misfolding and accumulation of this protein. Numerous studies have used rotenone and other toxins to induce mitochondrial dysfunction and monitor the accumulation of α-synuclein, despite the wealth of information that these studies provide they often do not reflect the subtleties of the slow accumulation of mitochondrial dysfunction within ageing SN neurons.Therefore, we investigated the relationship between mitochondria and aggregated α-synuclein, focussing on how these forms affect neurons with and without mitochondrial dysfunction. We wanted to understand how aggregated α-synuclein impacted on the survival of cells with mitochondrial dysfunction, to enable a deeper understanding of the effect of these two processes on neuronal survival. To investigate this we used cells with mutations in and partial inhibition of complexes I and IV.     

α-Synuclein Suppression by Targeted Small Interfering RNA in the Primate Substantia Nigra

The protein α-synuclein is involved in the pathogenesis of Parkinson''s disease and other neurodegenerative disorders. Its toxic potential appears to be enhanced by increased protein expression, providing a compelling rationale for therapeutic strategies aimed at reducing neuronal α-synuclein burden. Here, feasibility and safety of α-synuclein suppression were evaluated by treating monkeys with small interfering RNA (siRNA) directed against α-synuclein. The siRNA molecule was chemically modified to prevent degradation by exo- and endonucleases and directly infused into the left substantia nigra. Results compared levels of α-synuclein mRNA and protein in the infused (left) vs. untreated (right) hemisphere and revealed a significant 40–50% suppression of α-synuclein expression. These findings could not be attributable to non-specific effects of siRNA infusion since treatment of a separate set of animals with luciferase-targeting siRNA produced no changes in α-synuclein. Infusion with α-synuclein siRNA, while lowering α-synuclein expression, had no overt adverse consequences. In particular, it did not cause tissue inflammation and did not change (i) the number and phenotype of nigral dopaminergic neurons, and (ii) the concentrations of striatal dopamine and its metabolites. The data represent the first evidence of successful anti-α-synuclein intervention in the primate substantia nigra and support further development of RNA interference-based therapeutics.     

Role of alpha-synuclein in autophagy modulation of primary human T lymphocytes

It has been demonstrated that α-synuclein can aggregate and contribute to the pathogenesis of some neurodegenerative diseases and it is capable of hindering autophagy in neuronal cells. Here, we investigated the implication of α-synuclein in the autophagy process in primary human T lymphocytes. We provide evidence that: (i) knocking down of the α-synuclein gene resulted in increased autophagy, (ii) autophagy induction by energy deprivation was associated with a significant decrease of α-synuclein levels, (iii) autophagy inhibition by 3-methyladenine or by ATG5 knocking down led to a significant increase of α-synuclein levels, and (iv) autophagy impairment, constitutive in T lymphocytes from patients with systemic lupus erythematosus, was associated with abnormal accumulation of α-synuclein aggregates. These results suggest that α-synuclein could be considered as an autophagy-related marker of peripheral blood lymphocytes, potentially suitable for use in the clinical practice.     

Investigation of Intramolecular Dynamics and Conformations of α-, β- and γ-Synuclein

The synucleins are a family of natively unstructured proteins consisting of α-, β-, and γ-synuclein which are primarily expressed in neurons. They have been linked to a wide variety of pathologies, including neurological disorders, such as Parkinson’s disease (α-synuclein) and dementia with Lewy bodies (α- and β-synuclein), as well as various types of cancers (γ-synuclein). Self-association is a key pathological feature of many of these disorders, with α-synuclein having the highest propensity to form aggregates, while β-synuclein is the least prone. Here, we used a combination of fluorescence correlation spectroscopy and single molecule Förster resonance energy transfer to compare the intrinsic dynamics of different regions of all three synuclein proteins to investigate any correlation with putative functional or dysfunctional interactions. Despite a relatively high degree of sequence homology, we find that individual regions sample a broad range of diffusion coefficients, differing by almost a factor of four. At low pH, a condition that accelerates aggregation of α-synuclein, on average smaller diffusion coefficients are measured, supporting a hypothesis that slower intrachain dynamics may be correlated with self-association. Moreover, there is a surprising inverse correlation between dynamics and bulkiness of the segments. Aside from this observation, we could not discern any clear relationship between the physico-chemical properties of the constructs and their intrinsic dynamics. This work suggests that while protein dynamics may play a role in modulating self-association or interactions with other binding partners, other factors, particularly the local cellular environment, may be more important.     

Prion infection promotes extensive accumulation of α-synuclein in aged human α-synuclein transgenic mice

In neurodegenerative disorders of the aging population, misfolded proteins, such as PrP^Sc, α-synuclein, amyloid β protein and tau, can interact resulting in enhanced aggregation, cross seeding and accelerated disease progression. Previous reports have shown that in Creutzfeldt-Jakob disease and scrapie, α-synuclein accumulates near PrP^Sc deposits. However, it is unclear if pre-existing human α-synuclein aggregates modified prion disease pathogenesis, or if PrP^Sc exacerbates the α-synuclein pathology. Here, we inoculated infectious prions into aged α-synuclein transgenic (tg) and non-transgenic littermate control mice by the intracerebral route. Remarkably, inoculation of RML and mNS prions into α-synuclein tg mice resulted in more extensive and abundant intraneuronal and synaptic α-synuclein accumulation. In addition, infectious prions led to the formation of perineuronal α-synuclein deposits with a neuritic plaque-like appearance. Prion pathology was unmodified by the presence of α-synuclein. However, with the mNS prion strain there was a modest but significant acceleration in the time to terminal prion disease in mice having α-synuclein aggregates as compared with non-tg mice. Taken together, these studies support the notion that PrP^Sc directly or indirectly promotes α-synuclein pathology.     

Geometrical Membrane Curvature as an Allosteric Regulator of Membrane Protein Structure and Function

Transmembrane proteins are embedded in cellular membranes of varied lipid composition and geometrical curvature. Here, we studied for the first time the allosteric effect of geometrical membrane curvature on transmembrane protein structure and function. We used single-channel optical analysis of the prototypic transmembrane β-barrel α-hemolysin (α-HL) reconstituted on immobilized single small unilamellar liposomes of different diameter and therefore curvature. Our data demonstrate that physiologically abundant geometrical membrane curvatures can enforce a dramatic allosteric regulation (1000-fold inhibition) of α-HL permeability. High membrane curvatures (1/diameter ∼1/40 nm⁻¹) compressed the effective pore diameter of α-HL from 14.2 ± 0.8 Å to 11.4 ± 0.6 Å. This reduction in effective pore area (∼40%) when combined with the area compressibility of α-HL revealed an effective membrane tension of ∼50 mN/m and a curvature-imposed protein deformation energy of ∼7 k_BT. Such substantial energies have been shown to conformationally activate, or unfold, β-barrel and α-helical transmembrane proteins, suggesting that membrane curvature could likely regulate allosterically the structure and function of transmembrane proteins in general.     

A Highlights from MBoC Selection: Drosophila Histone Deacetylase 6 Protects Dopaminergic Neurons against α-Synuclein Toxicity by Promoting Inclusion Formation

Parkinson''s disease (PD) is associated with progressive degeneration of dopaminergic (DA) neurons. We report for the first time that the Drosophila histone deacetylase 6 (dHDAC6) plays a critical role in the protection of DA neurons and the formation of α-synuclein inclusions by using a Drosophila PD model constructed by ectopic expression of human α-synuclein. Depletion of dHDAC6 significantly enhances the effects caused by ectopic expression of α-synuclein, namely, loss of DA neurons, retinal degeneration, and locomotor dysfunction. Expression of α-synuclein in the DA neurons leads to fewer inclusions in the brains of dHDAC6 mutant flies than in wild-type flies. Conversely, overexpression of dHDAC6 is able to suppress the α-synuclein–induced DA neuron loss and retinal degeneration and promote inclusion formation. Furthermore, mutation of dHDAC6 reinforces the accumulation of oligomers that are suggested to be a toxic form of α-synuclein. We propose that α-synuclein inclusion formation in the presence of dHDAC6 protects DA neurons from being damaged by oligomers, which may uncover a common mechanism for synucleinopathies.     

Elevated Alpha-Synuclein Impairs Innate Immune Cell Function and Provides a Potential Peripheral Biomarker for Parkinson's Disease

Alpha-synuclein protein is strongly implicated in the pathogenesis Parkinson''s disease. Increased expression of α-synuclein due to genetic multiplication or point mutations leads to early onset disease. While α-synuclein is known to modulate membrane vesicle dynamics, it is not clear if this activity is involved in the pathogenic process or if measurable physiological effects of α-synuclein over-expression or mutation exist in vivo. Macrophages and microglia isolated from BAC α-synuclein transgenic mice, which overexpress α-synuclein under regulation of its own promoter, express α-synuclein and exhibit impaired cytokine release and phagocytosis. These processes were affected in vivo as well, both in peritoneal macrophages and microglia in the CNS. Extending these findings to humans, we found similar results with monocytes and fibroblasts isolated from idiopathic or familial Parkinson''s disease patients compared to age-matched controls. In summary, this paper provides 1) a new animal model to measure α-synuclein dysfunction; 2) a cellular system to measure synchronized mobilization of α-synuclein and its functional interactions; 3) observations regarding a potential role for innate immune cell function in the development and progression of Parkinson''s disease and other human synucleinopathies; 4) putative peripheral biomarkers to study and track these processes in human subjects. While altered neuronal function is a primary issue in PD, the widespread consequence of abnormal α-synuclein expression in other cell types, including immune cells, could play an important role in the neurodegenerative progression of PD and other synucleinopathies. Moreover, increased α-synuclein and altered phagocytosis may provide a useful biomarker for human PD.