Mutant Alpha-synuclein Research Articles

2.311 Microglial NADPH oxidase and macrophage antigen-1 receptor are essential to the enhanced dopaminergic neurodegeneration elicited by A30P and A53T mutant alpha-synuclein

2.311 Microglial NADPH oxidase and macrophage antigen-1 receptor are essential to the enhanced dopaminergic neurodegeneration elicited by A30P and A53T mutant alpha-synuclein

α-synuclein acts in the nucleus to inhibit histone acetylation and promote neurotoxicity

Alpha-synuclein is a neuronal protein implicated genetically in Parkinson's disease. alpha-synuclein localizes to the nucleus and presynaptic nerve terminals. Here we show that alpha-synuclein mediates neurotoxicity in the nucleus. Targeting of alpha-synuclein to the nucleus promotes toxicity, whereas cytoplasmic sequestration is protective in both cell culture and transgenic Drosophila. Toxicity of alpha-synuclein can be rescued by administration of histone deacetylase inhibitors in both cell culture and transgenic flies. Alpha-synuclein binds directly to histones, reduces the level of acetylated histone H3 in cultured cells and inhibits acetylation in histone acetyltransferase assays. Alpha-synuclein mutations that cause familial Parkinson's disease, A30P and A53T, exhibit increased nuclear targeting in cell culture. These findings implicate nuclear alpha-synuclein in promoting nigrostriatal degeneration in Parkinson's disease and encourage exploration of histone deacetylase inhibitors as potential therapies for the disorder.

α-Synuclein is colocalized with 14-3-3 and synphilin-1 in A53T transgenic mice

alpha-Synuclein is a major constituent of Lewy bodies, the neuropathological hallmark of Parkinson's disease (PD). Three types of alpha-synuclein mutations, A53T, A30P, and E46K, have been reported in familial PD. Wild-type alpha-synuclein accumulates at high concentrations in Lewy bodies, and this process is accelerated with mutated A53T alpha-synuclein. The accumulation of alpha-synuclein is thought to be toxic, and causes neuronal death when alpha-synuclein aggregates into protofibrils and fibrils. Lewy bodies contain not only alpha-synuclein, but also other proteins including 14-3-3 proteins and synphilin-1. 14-3-3 Proteins exist mainly as dimers and are related to intracellular signal transduction pathways. Synphilin-1 is known to interact with alpha-synuclein, promoting the formation of cytoplasmic inclusions like Lewy bodies in vitro. To investigate the colocalization of alpha-synuclein, synphilin-1, and 14-3-3 proteins, we performed immunohistochemical studies on alpha-synuclein, 14-3-3 proteins, and synphilin-1 in the brain and spinal cord of A53T transgenic mice. In homozygous mouse brains, alpha-synuclein immunoreactivity was observed in the neuronal somata and processes in the medial part of the brainstem, deep cerebellar nuclei, and spinal cord. The distribution of 14-3-3 proteins and synphilin-1 immunoreactivity was similar to that of alpha-synuclein in the homozygous mice. Double immunofluorescent staining showed that alpha-synuclein and synphilin-1 or 14-3-3 proteins were colocalized in the pons and spinal cord. These results indicate that the accumulation of mutant alpha-synuclein occurs in association with 14-3-3 proteins and synphilin-1, and may cause the sequestration of important proteins including 14-3-3 proteins and synphilin-1. The sequestration and subsequent decrease in 14-3-3 proteins and synphilin-1 levels may account for neuronal cell death.

Alpha‐synuclein inhibits aromatic amino acid decarboxylase activity in dopaminergic cells

Alpha-synuclein is a presynaptic protein strongly implicated in Parkinson's disease (PD). Because dopamine neurons are invariably compromised during pathogenesis in PD, we have been exploring the functions of alpha-synuclein with particular relevance to dopaminergic neuronal cells. We previously discovered reduced tyrosine hydroxylase (TH) activity and minimal dopamine synthesis in stably-transfected MN9D cells overexpressing either wild-type or A53T mutant (alanine to threonine at amino acid 53) alpha-synuclein. TH, the rate-limiting enzyme in dopamine synthesis, converts tyrosine to l-dihydroxyphenylalanine (L-DOPA), which is then converted to dopamine by the enzyme, aromatic amino acid decarboxylase (AADC). We confirmed an interaction between alpha-synuclein and AADC in striatum. We then sought to determine whether wild-type or A53T mutant alpha-synuclein might have affected AADC activity in dopaminergic cells. Using HPLC with electrochemical detection, we measured dopamine and related catechols after L-DOPA treatments to bypass the TH step. We discovered that while alpha-synuclein did not reduce AADC protein levels, it significantly reduced AADC activity and phosphorylation in our cells. These novel findings further support a role for alpha-synuclein in dopamine homeostasis and may explain, at least in part, the selective vulnerability of dopamine neurons that occurs in PD.

Abnormal compartmentalization of norepinephrine in mouse dentate gyrus in α‐synuclein knockout and A30P transgenic mice

In the dentate gyrus of the mouse hippocampus, presynaptic recruitment of norepinephrine in response to repeated-burst stimulation can be described in terms of an interaction between storage and readily releasable pools. The dynamics of this interaction depends on neuronal activity (bursting), so that the higher the demand for norepinephrine, the faster it is delivered from the storage pool. We also found that alpha-synuclein, a presynaptic protein that plays a crucial role in dopamine compartmentalization in the striatum, is also involved in the compartmentalization of norepinephrine in the dentate gyrus. Experiments in transgenic mice with modified or absent alpha-synuclein revealed that the familial Parkinson's disease-linked alpha-synuclein mutation A30P can cause selective changes in the function of noradrenergic terminals. Addition of mutated human alpha-synuclein abolished the normal norepinephrine mobilization. There were no compensatory mechanisms available in the norepinephrine presynaptic terminals. In contrast, deletion of mouse alpha-synuclein is compensated for by increased vesicle transport from the storage pool. The effects are essentially the same as previously reported for dopaminergic terminals in the striatum, indicating that the important role of alpha-synuclein in neurotransmitter mobilization is not limited to dopaminergic terminals.

P2-038: Membrane perturbation caused by mutant alpha-synuclein

P2-038: Membrane perturbation caused by mutant alpha-synuclein

Selective loss of dopaminergic neurons and formation of Lewy body‐like aggregations in α‐synuclein transgenic fly neuronal cultures

A key pathological feature of Parkinson's disease (PD) is the selective loss of dopaminergic neurons accompanied by the formation of Lewy bodies (LB). Given the complex nature of the disease, it is imperative to develop a model system suitable for molecular and cellular manipulation in order to study the mechanisms underlying the pathogenesis of PD. Here, we report that a new in vitro model of PD has been developed by using Drosophila melanogaster primary neuronal cultures expressing a human mutant alpha-synuclein (alpha-Syn; A30P). The selective loss of dopaminergic (DA) neurons was observed when alpha-Syn was pan-neuronally expressed while non-dopaminergic neurons (e.g. GABAergic) were not influenced. This degeneration was also observed even when alpha-Syn was specifically expressed in DA neurons, demonstrating alpha-Syn toxicity is DA cell-autonomous. In all experiments, cultures 5 days or older showed clear degeneration of DA neurons whereas this degeneration was not significant in 3-day-old cultures. In addition, there were intracellular aggregations in 5-day or older alpha-Syn neurons that were recognized by anti-alpha-Syn or ubiquitin antibodies, demonstrating the formation of LB-like inclusions. By contrast, no such aggregations were found in 3-day-old neurons. The results demonstrate that mutated human alpha-Syn expressed in Drosophila primary neuronal cultures causes the selective loss of DA neurons and the formation of cellular aggregations. Therefore, this is one of the first in vitro models recapitulating two important cellular features of PD and will be useful in examining mechanisms underlying selective neurodegeneration mediated by alpha-Syn.

Transduced Tat-α-Synuclein Protects against Oxidative Stress In vitro and In vivo

Parkinson's disease (PD) is a common neurodegenerative disorder and is characterized by the progressive loss of dopaminergic neurons in the substantia nigra. Although many studies showed that the aggregation of alpha-synuclein might be involved in the pathogenesis of PD, its protective properties against oxidative stress remain to be elucidated. In this study, human wild type and mutant alpha-synuclein genes were fused with a gene fragment encoding the nine amino acid transactivator of transcription (Tat) protein transduction domain of HIV-1 in a bacterial expression vector to produce a genetic in-frame WT Tat-alpha-synuclein (wild type) and mutant Tat-alpha-synucleins (mutants; A30P and A53T), respectively, and we investigated the protective effects of wild type and mutant Tat-alpha-synucleins in vitro and in vivo. WT Tat-alpha-synuclein rapidly transduced into an astrocyte cells and protected the cells against paraquat induced cell death. However, mutant Tat-alpha-synucleins did not protect at all. In the mice models exposed to the herbicide paraquat, the WT Tat-alpha-synuclein completely protected against dopaminergic neuronal cell death, whereas mutants failed in protecting against oxidative stress. We found that these protective effects were characterized by increasing the expression level of heat shock protein 70 (HSP70) in the neuronal cells and this expression level was dependent on the concentration of transduced WT Tat-alpha-synuclein. These results suggest that transduced Tat-alpha-synuclein might protect cell death from oxidative stress by increasing the expression level of HSP70 in vitro and in vivo and this may be of potential therapeutic benefit in the pathogenesis of PD.

Embryonic stem cell‐derived neuron models of Parkinson's disease exhibit delayed neuronal death

Establishment of a Parkinson's disease (PD) neuron model was attempted with mouse embryonic stem (ES) cells. ES cell lines over-expressing mouse nuclear receptor-related 1 (Nurr1), together with human wild-type and alanine 30 --> proline (A30P) and alanine 53 --> threonine (A53T) mutant alpha-synuclein were established and subjected to differentiation into dopaminergic neurons. The ES cell-derived dopaminergic neurons expressing wild-type or mutant alpha-synuclein exhibited the fundamental characteristics consistent with dopaminergic neurons in the substantia nigra. The ES cell-derived PD model neurons exhibited increased susceptibility to oxidative stress, proteasome inhibition, and mitochondrial inhibition. Cell viability of PD model neurons and the control neurons was similar until 28 days after differentiation. Nonetheless, after that time, PD model neurons gradually began to undergo neuronal death over the course of 1 month, showing cytoplasmic aggregate formation and an increase of insoluble alpha-synuclein protein. Such delayed neuronal death was observed in a mutant alpha-synuclein protein level-dependent manner, which was slightly inhibited by a c-jun N-terminal kinase inhibitor and a caspase inhibitor. Such cell death was not observed when the same ES cell lines were differentiated into oligodendrocytes. The ES cell-derived PD model neurons are considered as prospective candidates for a new prototype modelling PD that would allow better investigation of the underlying neurodegenerative pathophysiology.

Etiology of Parkinson’s disease

The etiology of Parkinson's disease (PD) has long been thought to involve both genetic and environmental factors, but until recently there has been no direct evidence to support either one as a causative factor. However, in the past 8 years six different genes have been identified as causing familial PD. Together, they support the notion that common pathogenetic mechanisms exist across the etiologic spectrum of PD. Specifically, mutations in alpha-synuclein, parkin, UCHL1, DJ1, PINK1, and LRRK2 cause PD, with a Mendelian pattern of inheritance. DJ1 and PINK1 are mitochondrial proteins and overexpression of alpha-synuclein and parkin induce mitochondrial defects. These same proteins are involved in the response to oxidative stress and affect proteasomal function. In contrast, few environmental factors have been characterized. Nevertheless, those toxins that have been demonstrated to have the ability to cause nigrostriatal cell death appear to interact by interfering with mitochondrial function, inducing oxidative stress, and modifying proteasomal function. Therefore, common themes are beginning to emerge in the etiopathogenesis of PD. This bodes well for research focused on the development of treatments that will modify the course of PD.

Proteasomal inhibition hypersensitizes differentiated neuroblastoma cells to oxidative damage

Parkinson's disease (PD) is a multifactorial disease caused by both genetic and environmental factors. Alpha-synuclein is of particular interest in PD since it is a major component of Lewy bodies and mutations in the alpha-synuclein gene were identified in familial PD. Oxidative stress and proteasomal dysfunction are implicated in the pathogenesis of PD but their interactions as well as their effect on aggregates formation are not yet clear. We therefore examined the roles of oxidative stress and proteasomal inhibition on protein aggregates induction in naïve and neuronally differentiated neuroblastoma SH-SY5Y cells. Neuroblastoma cells were stably transfected with wild type (WT) and A53T mutant alpha-synuclein. Naïve and transfected cells were exposed to oxidative stress induced by rotenone, SIN-I, FeCl 2, and to proteasomal inhibition by lactacystin. Proteasomal inhibition caused a dose-dependent decrease in viability and induced protein aggregates formation containing alpha-synuclein and ubiquitin. Proteasomal inhibition induced significantly increased alpha-synuclein aggregation in cells expressing mutant alpha-synuclein. Exposure to reactive oxygen species (ROS) combined with proteasomal inhibition increased aggregates formation. Inclusion body formation and cell death of differentiated neuroblastoma cells overexpressing alpha-synuclein can serve as a valuable model for elucidating the molecular components that cause neurodegeneration in PD as well as evaluating pharmacological interventions.

Plasma membrane ion permeability induced by mutant α‐synuclein contributes to the degeneration of neural cells

Mutations in alpha-synuclein cause some cases of familial Parkinson's disease (PD), but the mechanism by which alpha-synuclein promotes degeneration of dopamine-producing neurons is unknown. We report that human neural cells expressing mutant alpha-synuclein (A30P and A53T) have higher plasma membrane ion permeability. The higher ion permeability caused by mutant alpha-synuclein would be because of relatively large pores through which most cations can pass non-selectively. Both the basal level of [Ca2+]i and the Ca2+ response to membrane depolarization are greater in cells expressing mutant alpha-synuclein. The membrane permeable Ca2+ chelator BAPTA-AM significantly protected the cells against oxidative stress, whereas neither L-type (nifedipine) nor N-type (omega-conotoxin-GVIA) Ca2+ channel blockers protected the cells. These findings suggest that the high membrane ion permeability caused by mutant alpha-synuclein may contribute to the degeneration of neurons in PD.

Association of α-Synuclein and Mutants with Lipid Membranes: Spin-Label ESR and Polarized IR

Alpha-synuclein is a presynaptic protein, the A53T and A30P mutants of which are linked independently to early-onset familial Parkinson's disease. The association of wild-type alpha-synuclein with lipid membranes was characterized previously by electron spin resonance (ESR) spectroscopy with spin-labeled lipids [Ramakrishnan, M., Jensen, P. H., and Marsh, D. (2003) Biochemistry 42, 12919-12926]. Here, we study the interaction of the A53T and A30P alpha-synuclein mutants and a truncated form that lacks the acidic C-terminal domain with phosphatidylglycerol bilayer membranes, using anionic phospholipid spin labels. The strength of the interaction with phosphatidylglycerol membranes lies in the order: wild type approximately truncated > A53T > A30P > fibrils approximately 0, and only the truncated form interacts with phosphatidylcholine membranes. The selectivity of the interaction of the mutant alpha-synucleins with different spin-labeled lipid species is reduced considerably, relative to the wild-type protein, whereas that of the truncated protein is increased. Polarized infrared (IR) spectroscopy is used to study the interactions of the wild-type and truncated proteins with aligned lipid membranes and additionally to characterize the fibrillar form. Wild-type alpha-synuclein is natively unfolded in solution and acquires secondary structure upon binding to membranes containing phosphatidylglycerol. Up to 30-40% of the amide I band intensity of the membrane-bound wild-type and truncated proteins is attributable to beta-sheet structure, at the surface densities used for IR spectroscopy. The remainder is alpha-helix and residual unordered structure. Fibrillar alpha-synuclein contains 62% antiparallel beta-sheet and is oriented on the substrate surface but does not interact with deposited lipid membranes. The beta-sheet secondary-structural elements of the wild-type and truncated proteins are partially oriented on the surface of membranes with which they interact.

Leucine-rich repeat kinase 2 (LRRK2) interacts with parkin, and mutant LRRK2 induces neuronal degeneration.

Parkinson's disease (PD) is a disorder of movement, cognition, and emotion, and it is characterized pathologically by neuronal degeneration with Lewy bodies, which are cytoplasmic inclusion bodies containing deposits of aggregated proteins. Most PD cases appear to be sporadic, but genetic forms of the disease, caused by mutations in alpha-synuclein, parkin, and other genes, have helped elucidate pathogenesis. Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal-dominant Parkinsonism with clinical features of PD and with pleomorphic pathology including deposits of aggregated protein. To study expression and interactions of LRRK2, we synthesized cDNAs and generated expression constructs coding for human WT and mutant LRRK2 proteins. Expression of full-length LRRK2 in cells in culture suggests that the protein is predominately cytoplasmic, as is endogenous protein by subcellular fractionation. Using coimmunoprecipitation, we find that LRRK2, expressed in cells in culture, interacts with parkin but not with alpha-synuclein, DJ-1, or tau. A small proportion of the cells overexpressing LRRK2 contain protein aggregates, and this proportion is greatly increased by coexpression of parkin. In addition, parkin increases ubiquitination of aggregated protein. Also, mutant LRRK2 causes neuronal degeneration in both SH-SY5Y cells and primary neurons. This cell model may be useful for studies of PD cellular pathogenesis and therapeutics. These findings suggest a gain-of-function mechanism in the pathogenesis of LRRK2-linked PD and suggest that LRRK2 may be involved in a pathogenic pathway with other PD-related proteins such as parkin, which may help illuminate both familial and sporadic PD.

The alpha-synuclein mutation E46K promotes aggregation in cultured cells

Parkinson’s disease (PD) is characterized by the polymerization of wild-type (WT) or mutant alpha-synuclein (AS) into aggregates and fibrils, which are observed as Lewy bodies (LBs) and Lewy neurites (LNs) in PD patients. However, inability to demonstrate aggregation in many cell culture systems is a major drawback for effective in vitro modeling of AS aggregation. Utilizing PCR-based cloning approach, we generated A30P, A53T, and the recently reported E46K encoding mutation in the KTKEGV repeat region of AS gene. While cloning E46K mutant, a glycine deletion mutation (E46KΔG) adjacent to the intended lysine mutation was serendipitously generated. Expression of mutant constructs and green fluorescent protein (GFP)-tagged mutant constructs in catecholaminergic SH-SY5Y (5Y) cells revealed 40% of AS-E46KΔG and 18% of AS-E46K transfected cells formed aggregates as compared to 12% in AS-A53T, 6% in AS-WT, and 2% in AS-A30P transfected cells. Western blot analysis demonstrated the formation of high molecular weight AS aggregates. Electron microscopic analysis of 5Y cells expressing the E46K and E46KΔG mutants demonstrated two distinct kinds of inclusions: Type I, which showed dense granular profile; and Type II, which were largely membranous vacuolar inclusions without granular material. These two inclusions are reminiscent of Lewy bodies and pale bodies observed in PD postmortem brain samples. Our results demonstrate that mutations in 4th KTKEGV repeat lead to higher propensity of aggregation of AS compared to other mutants.

Dopamine-related and caspase-independent apoptosis in dopaminergic neurons induced by overexpression of human wild type or mutant α-synuclein

Human wild type (WT) and mutant alpha-synuclein (alpha-syn) genes were overexpressed using a Tet-on expression system in stably transfected dopaminergic MN9D cells. Their overexpression induced caspase-independent and dopamine-related apoptosis not rescued by general caspase inhibitor Z-VAD-FMK. While apoptosis due to overexpression of WT alpha-syn was completely abrogated by a specific tyrosine hydroxylase (TH) inhibitor, alpha-methyl-p-tyrosine (alpha-MT), the inhibitor only partially rescued apoptosis caused by overexpression of alpha-syn mutants. In addition, overexpression of mutants enhanced the toxicity of 1-methyl-4-phenylpyridinium (MPP+) and 6-hydroxyldopamine (6-OHDA) to MN9D cells, whereas overexpression of WT protected MN9D cells against MPP+ toxicity, but not against 6-OHDA. We conclude that WT alpha-syn is beneficial to dopaminergic neurons but its overexpression in the presence of endogenous dopamine makes it a potential threat to the cells. In contrast, mutant alpha-syn not only caused the loss of WT protective function but also the gain-of-toxicity which becomes more serious in the presence of dopamine and neurotoxins.

α-Synuclein Cooperates with CSPα in Preventing Neurodegeneration

Alpha-synuclein and cysteine-string protein-alpha (CSPalpha) are abundant synaptic vesicle proteins independently linked to neurodegeneration. Dominantly inherited mutations in alpha-synuclein cause Parkinson's disease, but the physiological role of alpha-synuclein remains unknown. Deletion of CSPalpha produces rapidly progressive neurodegeneration in mice, presumably because the cochaperone function of CSPalpha is essential for neuronal survival. Here, we report the surprising finding that transgenic expression of alpha-synuclein abolishes the lethality and neurodegeneration caused by deletion of CSPalpha. Conversely, ablation of endogenous synucleins exacerbates these phenotypes. Deletion of CSPalpha inhibits SNARE complex assembly; transgenic alpha-synuclein ameliorates this inhibition. In preventing neurodegeneration in CSPalpha-deficient mice, alpha-synuclein does not simply substitute for CSPalpha but acts by a downstream mechanism that requires phospholipid binding by alpha-synuclein. These observations reveal a powerful in vivo activity of alpha-synuclein in protecting nerve terminals against injury and suggest that this activity operates in conjunction with CSPalpha and SNARE proteins on the presynaptic membrane interface.

Endoplasmic reticulum stress and mitochondrial cell death pathways mediate A53T mutant alpha-synuclein-induced toxicity

Parkinson's disease (PD) is a neurodegenerative movement disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. Alpha-synuclein is a major component of Lewy bodies in sporadic PD, and mutations in alpha-synuclein cause autosomal-dominant hereditary PD. Here, we generated A53T mutant alpha-synuclein-inducible PC12 cell lines using the Tet-off regulatory system. Inducing expression of A53T alpha-synuclein in differentiated PC12 cells decreased proteasome activity, increased the intracellular ROS level and caused up to approximately 40% cell death, which was accompanied by mitochondrial cytochrome C release and elevation of caspase-9 and -3 activities. Cell death was partially blocked by cyclosporine A [an inhibitor of the mitochondrial permeability transition (MPT) process], z-VAD (a pan-caspase inhibitor) and inhibitors of caspase-9 and -3 but not by a caspase-8 inhibitor. Furthermore, induction of A53T alpha-synuclein increased endoplasmic reticulum (ER) stress and elevated caspase-12 activity. RNA interference to knock down caspase-12 levels or salubrinal (an ER stress inhibitor) partially protected against cell death and further reduced A53T toxicity after treatment with z-VAD. Our results indicate that both ER stress and mitochondrial dysfunction contribute to A53T alpha-synuclein-induced cell death. This study sheds light into the pathogenesis of alpha-synuclein cellular toxicity in PD and provides a cell model for screening PD therapeutic agents.

Differential proteome analysis of brain from alpha-synuclein mutant mice

Differential proteome analysis of brain from alpha-synuclein mutant mice

PA700, the regulatory complex of the 26S proteasome, interferes with α‐synuclein assembly

Parkinson's disease is characterized by the loss of dopaminergic neurons in the nigrostriatal pathway accompanied by the presence of intracellular cytoplasmic inclusions, termed Lewy bodies. Fibrillized alpha-synuclein forms the major component of Lewy bodies. We reported a specific interaction between rat alpha-synuclein and tat binding protein 1, a subunit of PA700, the regulatory complex of the 26S proteasome. It has been demonstrated that PA700 prevents the aggregation of misfolded, nonubiquinated substrates. In this study, we examine the effect of PA700 on the aggregation of wild-type and A53T mutant alpha-synuclein. PA700 inhibits both wild-type and A53T alpha-synuclein fibril formation as measured by Thioflavin T fluorescence. Using size exclusion chromatography, we present evidence for a stable PA700-alpha-synuclein complex. Sedimentation analyses reveal that PA700 sequesters alpha-synuclein in an assembly incompetent form. Analysis of the morphology of wild-type and A53T alpha-synuclein aggregates during the course of fibrillization by electron microscopy demonstrate the formation of amyloid-like fibrils. Secondary structure analyses of wild-type and A53T alpha-synuclein assembled in the presence of PA700 revealed a decrease in the overall amount of assembled alpha-synuclein with no significant change in protein conformation. Thus, PA700 acts on alpha-synuclein assembly and not on the structure of fibrils. We hypothesize that PA700 sequesters alpha-synuclein oligomeric species that are the precursors of the fibrillar form of the protein, thus preventing its assembly into fibrils.