?-synuclein Induces Mitochondrial Dysfunction through Spectrin and the Actin Cytoskeleton.
ABSTRACT: Genetics and neuropathology strongly link ?-synuclein aggregation and neurotoxicity to the pathogenesis of Parkinson's disease and related ?-synucleinopathies. Here we describe a new Drosophila model of ?-synucleinopathy based on widespread expression of wild-type human ?-synuclein, which shows robust neurodegeneration, early-onset locomotor deficits, and abundant ?-synuclein aggregation. We use results of forward genetic screening and genetic analysis in our new model to demonstrate that ?-synuclein expression promotes reorganization of the actin filament network and consequent mitochondrial dysfunction through altered Drp1 localization. Similar changes are present in a mouse ?-synucleinopathy model and in postmortem brain tissue from patients with ?-synucleinopathy. Importantly, we provide evidence that the interaction of ?-synuclein with spectrin initiates pathological alteration of the actin cytoskeleton and downstream neurotoxicity. These findings suggest new therapeutic approaches for ?-synuclein induced neurodegeneration.
Project description:Aggregation of ?-synuclein contributes to the formation of Lewy bodies and neurites, the pathologic hallmarks of Parkinson disease (PD) and ?-synucleinopathies. Although a number of human mutations have been identified in familial PD, the mechanisms that promote ?-synuclein accumulation and toxicity are poorly understood. Here, we report that hyperactivity of the nonreceptor tyrosine kinase c-Abl critically regulates ?-synuclein-induced neuropathology. In mice expressing a human ?-synucleinopathy-associated mutation (hA53T?-syn mice), deletion of the gene encoding c-Abl reduced ?-synuclein aggregation, neuropathology, and neurobehavioral deficits. Conversely, overexpression of constitutively active c-Abl in hA53T?-syn mice accelerated ?-synuclein aggregation, neuropathology, and neurobehavioral deficits. Moreover, c-Abl activation led to an age-dependent increase in phosphotyrosine 39 ?-synuclein. In human postmortem samples, there was an accumulation of phosphotyrosine 39 ?-synuclein in brain tissues and Lewy bodies of PD patients compared with age-matched controls. Furthermore, in vitro studies show that c-Abl phosphorylation of ?-synuclein at tyrosine 39 enhances ?-synuclein aggregation. Taken together, this work establishes a critical role for c-Abl in ?-synuclein-induced neurodegeneration and demonstrates that selective inhibition of c-Abl may be neuroprotective. This study further indicates that phosphotyrosine 39 ?-synuclein is a potential disease indicator for PD and related ?-synucleinopathies.
Project description:An increase in ?-synuclein levels due to gene duplications/triplications or impaired degradation is sufficient to trigger its aggregation and cause familial Parkinson disease (PD). Therefore, lowering ?-synuclein levels represents a viable therapeutic strategy for the treatment of PD and related synucleinopathies. Here, we report that Polo-like kinase 2 (PLK2), an enzyme up-regulated in synucleinopathy-diseased brains, interacts with, phosphorylates and enhances ?-synuclein autophagic degradation in a kinase activity-dependent manner. PLK2-mediated degradation of ?-synuclein requires both phosphorylation at S129 and PLK2/?-synuclein complex formation. In a rat genetic model of PD, PLK2 overexpression reduces intraneuronal human ?-synuclein accumulation, suppresses dopaminergic neurodegeneration, and reverses hemiparkinsonian motor impairments induced by ?-synuclein overexpression. This PLK2-mediated neuroprotective effect is also dependent on PLK2 activity and ?-synuclein phosphorylation. Collectively, our findings demonstrate that PLK2 is a previously undescribed regulator of ?-synuclein turnover and that modulating its kinase activity could be a viable target for the treatment of synucleinopathies.
Project description:It is necessary to develop an understanding of the specific mechanisms involved in alpha-synuclein aggregation and propagation to develop disease modifying therapies for age-related synucleinopathies, including Parkinson's disease and Dementia with Lewy Bodies. To adequately address this question, we developed a new transgenic mouse model of synucleinopathy that expresses human A53T SynGFP under control of the mouse prion protein promoter. Our characterization of this mouse line demonstrates that it exhibits several distinct advantages over other, currently available, mouse models. This new model allows rigorous study of the initial location of Lewy pathology formation and propagation in the living brain, and strongly suggests that aggregation begins in axonal structures with retrograde propagation to the cell body. This model also shows expeditious development of alpha-synuclein pathology following induction with small, in vitro-generated alpha-synuclein pre-formed fibrils (PFFs), as well as accelerated cell death of inclusion-bearing cells. Using this model, we found that aggregated alpha-synuclein somatic inclusions developed first in neurons, but later showed a second wave of inclusion formation in astrocytes. Interestingly, astrocytes appear to survive much longer after inclusion formation than their neuronal counterparts. This model also allowed careful study of peripheral-to-central spread of Lewy pathology after PFF injection into the hind limb musculature. Our results clearly show evidence of progressive, retrograde trans-synaptic spread of Lewy pathology through known neuroanatomically connected pathways in the motor system. As such, we have developed a promising tool to understand the biology of neurodegeneration associated with alpha-synuclein aggregation and to discover new treatments capable of altering the neurodegenerative disease course of synucleinopathies.
Project description:The accumulation of misfolded a-synuclein is mechanistically linked to neurodegeneration in Parkinson's disease (PD) and other alpha-synucleinopathies. However, how alpha-synuclein causes neurodegeneration is unresolved. Several studies have supported the involvement of dynein, the major motor for retrograde axonal transport in alpha-synuclein-dependent neurodegeneration, especially in the nigrostriatal system. Therefore, we examined the nigrostriatal dyneins in transgenic mice that overexpress human A53T alpha-synuclein and recapitulate key features of a PD-like neuronal synucleinopathy. Age-matched nontransgenic littermates were used as controls. The results demonstrated that the protein level of dynein was decreased in the striatum, whereas it was elevated in the substantia nigra. Double immunostaining results revealed that the reduction in dynein level was associated with aggregation of A53T a-synuclein in the striatum. Furthermore, we performed a quantitative analysis of motor behaviors in A53T alpha-synuclein transgenic mice and controls using a modified open field test. We demonstrated that the protein level of dynein in the striatum was significantly correlated with the motor behaviors. Together, our data indicate that dynein changes in the nigrostriatal system of A53T alpha-synuclein transgenic mice may contribute to their severe movement disorder.
Project description:Synucleinopathies of the aging population are an heterogeneous group of neurological disorders that includes Parkinson's disease (PD) and dementia with Lewy bodies (DLB) and are characterized by the progressive accumulation of ?-synuclein in neuronal and glial cells. Toll-like receptor 2 (TLR2), a pattern recognition immune receptor, has been implicated in the pathogenesis of synucleinopathies because TLR2 is elevated in the brains of patients with PD and TLR2 is a mediator of the neurotoxic and pro-inflammatory effects of extracellular ?-synuclein aggregates. Therefore, blocking TLR2 might alleviate ?-synuclein pathological and functional effects. For this purpose, herein, we targeted TLR2 using a functional inhibitory antibody (anti-TLR2).Two different human ?-synuclein overexpressing transgenic mice were used in this study. ?-synuclein low expresser mouse (?-syn-tg, under the PDGF? promoter, D line) was stereotaxically injected with TLR2 overexpressing lentivirus to demonstrate that increment of TLR2 expression triggers neurotoxicity and neuroinflammation. ?-synuclein high expresser mouse (?-Syn-tg; under mThy1 promoter, Line 61) was administrated with anti-TLR2 to examine that functional inhibition of TLR2 ameliorates neuropathology and behavioral defect in the synucleinopathy animal model. In vitro ?-synuclein transmission live cell monitoring system was used to evaluate the role of TLR2 in ?-synuclein cell-to-cell transmission.We demonstrated that administration of anti-TLR2 alleviated ?-synuclein accumulation in neuronal and astroglial cells, neuroinflammation, neurodegeneration, and behavioral deficits in an ?-synuclein tg mouse model of PD/DLB. Moreover, in vitro studies with neuronal and astroglial cells showed that the neuroprotective effects of anti-TLR2 antibody were mediated by blocking the neuron-to-neuron and neuron-to-astrocyte ?-synuclein transmission which otherwise promotes NF?B dependent pro-inflammatory responses.This study proposes TLR2 immunotherapy as a novel therapeutic strategy for synucleinopathies of the aging population.
Project description:?-Synucleinopathies are neurodegenerative diseases that are characterized pathologically by ?-synuclein inclusions in neurons and glia. The pathologic contribution of glial ?-synuclein in these diseases is not well understood. Glial ?-synuclein may be of particular importance in multiple system atrophy (MSA), which is defined pathologically by glial cytoplasmic ?-synuclein inclusions. We have previously described Drosophila models of neuronal ?-synucleinopathy, which recapitulate key features of the human disorders. We have now expanded our model to express human ?-synuclein in glia. We demonstrate that expression of ?-synuclein in glia alone results in ?-synuclein aggregation, death of dopaminergic neurons, impaired locomotor function, and autonomic dysfunction. Furthermore, co-expression of ?-synuclein in both neurons and glia worsens these phenotypes as compared to expression of ?-synuclein in neurons alone. We identify unique transcriptomic signatures induced by glial as opposed to neuronal ?-synuclein. These results suggest that glial ?-synuclein may contribute to the burden of pathology in the ?-synucleinopathies through a cell type-specific transcriptional program. This new Drosophila model system enables further mechanistic studies dissecting the contribution of glial and neuronal ?-synuclein in vivo, potentially shedding light on mechanisms of disease that are especially relevant in MSA but also the ?-synucleinopathies more broadly.
Project description:?-Synuclein is a soluble protein that is present in abundance in the brain, though its normal function in the healthy brain is poorly defined. Intraneuronal inclusions of ?-synuclein, commonly referred to as Lewy pathology, are pathological hallmarks of a spectrum of neurodegenerative disorders referred to as ?-synucleinopathies. Though ?-synuclein is expressed predominantly in neurons, ?-synuclein aggregates in astrocytes are a common feature in these neurodegenerative diseases. How and why ?-synuclein ends up in the astrocytes and the consequences of this dysfunctional proteostasis in immune cells is a major area of research that can have far-reaching implications for future immunobiotherapies in ?-synucleinopathies. Accumulation of aggregated ?-synuclein can disrupt astrocyte function in general and, more importantly, can contribute to neurodegeneration in ?-synucleinopathies through various pathways. Here, we summarize our current knowledge on how astrocytic ?-synucleinopathy affects CNS function in health and disease and propose a model of neuroglial connectome altered by ?-synuclein proteostasis that might be amenable to immune-based therapies.
Project description:Mutations in the neuronal protein alpha-synuclein cause familial Parkinson disease. Phosphorylation of alpha-synuclein at serine 129 is prominent in Parkinson disease and influences alpha-synuclein neurotoxicity. Here we report that alpha-synuclein is also phosphorylated at tyrosine 125 in transgenic Drosophila expressing wild-type human alpha-synuclein and that this tyrosine phosphorylation protects from alpha-synuclein neurotoxicity in a Drosophila model of Parkinson disease. Western blot analysis of fly brain homogenates showed that levels of soluble oligomeric species of alpha-synuclein were increased by phosphorylation at serine 129 and decreased by tyrosine 125 phosphorylation. Tyrosine 125 phosphorylation diminished during the normal aging process in both humans and flies. Notably, cortical tissue from patients with the Parkinson disease-related synucleinopathy dementia with Lewy bodies showed less phosphorylation at tyrosine 125. Our findings suggest that alpha-synuclein neurotoxicity in Parkinson disease and related synucleinopathies may result from an imbalance between the detrimental, oligomer-promoting effect of serine 129 phosphorylation and a neuroprotective action of tyrosine 125 phosphorylation that inhibits toxic oligomer formation.
Project description:Accumulation of misfolded ?-synuclein (?S) is mechanistically linked to neurodegeneration in Parkinson's disease (PD) and other ?-synucleinopathies. However, how ?S causes neurodegeneration is unresolved. Because cellular accumulation of misfolded proteins can lead to endoplasmic reticulum stress/unfolded protein response (ERS/UPR), chronic ERS could contribute to neurodegeneration in ?-synucleinopathy. Using the A53T mutant human ?S transgenic (A53T?S Tg) mouse model of ?-synucleinopathy, we show that disease onset in the ?S Tg model is coincident with induction of ER chaperones in neurons exhibiting ?S pathology. However, the neuronal ER chaperone induction was not accompanied by the activation of phospho-eIF2?, indicating that ?-synucleinopathy is associated with abnormal UPR that could promote cell death. Induction of ERS/UPR was associated with increased levels of ER/microsomal (ER/M) associated ?S monomers and aggregates. Significantly, human PD cases also exhibit higher relative levels of ER/M ?S than the control cases. Moreover, ?S interacts with ER chaperones and overexpression of ?S sensitizes neuronal cells to ERS-induced toxicity, suggesting that ?S may have direct impact on ER function. This view is supported by the presence of ERS-activated caspase-12 and the accumulation of ER-associated polyubiquitin. More important, treatment with Salubrinal, an anti-ERS compound, significantly attenuates disease manifestations in both the A53T?S Tg mouse model and the adeno-associated virus-transduced rat model of A53T?S-dependent dopaminergic neurodegeneration. Our data indicate that the accumulation ?S within ER leads to chronic ER stress conditions that contribute to neurodegeneration in ?-synucleinopathies. Attenuating chronic ERS could be an effective therapy for PD and other ?-synucleinopathies.
Project description:BACKGROUND:?-Synuclein (aSyn) aggregation is thought to play a central role in neurodegenerative disorders termed synucleinopathies, including Parkinson's disease (PD). Mouse aSyn contains a threonine residue at position 53 that mimics the human familial PD substitution A53T, yet in contrast to A53T patients, mice show no evidence of aSyn neuropathology even after aging. Here, we studied the neurotoxicity of human A53T, mouse aSyn, and various human-mouse chimeras in cellular and in vivo models, as well as their biochemical properties relevant to aSyn pathobiology. METHODS:Primary midbrain cultures transduced with aSyn-encoding adenoviruses were analyzed immunocytochemically to determine relative dopaminergic neuron viability. Brain sections prepared from rats injected intranigrally with aSyn-encoding adeno-associated viruses were analyzed immunohistochemically to determine nigral dopaminergic neuron viability and striatal dopaminergic terminal density. Recombinant aSyn variants were characterized in terms of fibrillization rates by measuring thioflavin T fluorescence, fibril morphologies via electron microscopy and atomic force microscopy, and protein-lipid interactions by monitoring membrane-induced aSyn aggregation and aSyn-mediated vesicle disruption. Statistical tests consisted of ANOVA followed by Tukey's multiple comparisons post hoc test and the Kruskal-Wallis test followed by a Dunn's multiple comparisons test or a two-tailed Mann-Whitney test. RESULTS:Mouse aSyn was less neurotoxic than human aSyn A53T in cell culture and in rat midbrain, and data obtained for the chimeric variants indicated that the human-to-mouse substitutions D121G and N122S were at least partially responsible for this decrease in neurotoxicity. Human aSyn A53T and a chimeric variant with the human residues D and N at positions 121 and 122 (respectively) showed a greater propensity to undergo membrane-induced aggregation and to elicit vesicle disruption. Differences in neurotoxicity among the human, mouse, and chimeric aSyn variants correlated weakly with differences in fibrillization rate or fibril morphology. CONCLUSIONS:Mouse aSyn is less neurotoxic than the human A53T variant as a result of inhibitory effects of two C-terminal amino acid substitutions on membrane-induced aSyn aggregation and aSyn-mediated vesicle permeabilization. Our findings highlight the importance of membrane-induced self-assembly in aSyn neurotoxicity and suggest that inhibiting this process by targeting the C-terminal domain could slow neurodegeneration in PD and other synucleinopathy disorders.