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Alzheimer's disease is a prominent chronic neurodegenerative condition characterized by a gradual decline in memory leading to dementia. Growing evidence suggests that Alzheimer's disease is associated with accumulating various amyloid-ß oligomers in the brain, influenced by complex genetic and environmental factors. The memory and cognitive deficits observed during the prodromal and mild cognitive impairment phases of Alzheimer's disease are believed to primarily result from synaptic dysfunction. Throughout life, environmental factors can lead to enduring changes in gene expression and the emergence of brain disorders. These changes, known as epigenetic modifications, also play a crucial role in regulating the formation of synapses and their adaptability in response to neuronal activity. In this context, we highlight recent advances in understanding the roles played by key components of the epigenetic machinery, specifically DNA methylation, histone modification, and microRNAs, in the development of Alzheimer's disease, synaptic function, and activity-dependent synaptic plasticity. Moreover, we explore various strategies, including enriched environments, exposure to non-invasive brain stimulation, and the use of pharmacological agents, aimed at improving synaptic function and enhancing long-term potentiation, a process integral to epigenetic mechanisms. Lastly, we deliberate on the development of effective epigenetic agents and safe therapeutic approaches for managing Alzheimer's disease. We suggest that addressing Alzheimer's disease may require distinct tailored epigenetic drugs targeting different disease stages or pathways rather than relying on a single drug.
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The heterogeneity of protein-rich inclusions and its significance in neurodegeneration is poorly understood. Standard patient-derived iPSC models develop inclusions neither reproducibly nor in a reasonable time frame. Here, we developed screenable iPSC "inclusionopathy" models utilizing piggyBac or targeted transgenes to rapidly induce CNS cells that express aggregation-prone proteins at brain-like levels. Inclusions and their effects on cell survival were trackable at single-inclusion resolution. Exemplar cortical neuron α-synuclein inclusionopathy models were engineered through transgenic expression of α-synuclein mutant forms or exogenous seeding with fibrils. We identified multiple inclusion classes, including neuroprotective p62-positive inclusions versus dynamic and neurotoxic lipid-rich inclusions, both identified in patient brains. Fusion events between these inclusion subtypes altered neuronal survival. Proteome-scale α-synuclein genetic- and physical-interaction screens pinpointed candidate RNA-processing and actin-cytoskeleton-modulator proteins like RhoA whose sequestration into inclusions could enhance toxicity. These tractable CNS models should prove useful in functional genomic analysis and drug development for proteinopathies.
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α-Synuclein (αS) is an abundant presynaptic protein that regulates neurotransmission. It is also a key protein implicated in a broad class of neurodegenerative disorders termed synucleinopathies, including Parkinson's disease (PD) and Lewy body dementia (LBD). Pathological αS deposits in these diseases, Lewy bodies (LBs)/neurites (LNs), contain about 90% of αS in its phospho-serine129 (pS129) form. Therefore, pS129 is widely used as a surrogate marker of pathology. However, recent findings demonstrate that pS129 is also physiologically triggered by neuronal activity to positively regulate synaptic transmission. In this opinion article, we contrast the literature on pathological and physiological pS129, with a special focus on the latter. We emphasize that pS129 is ambiguous and knowledge about the context is necessary to correctly interpret changes in pS129.
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alfa-Sinucleína , alfa-Sinucleína/metabolismo , Humanos , Fosforilação/fisiologia , Animais , Serina/metabolismo , Doença de Parkinson/metabolismo , Transmissão Sináptica/fisiologia , Sinucleinopatias/metabolismoRESUMO
Alpha-synuclein (αS)-rich Lewy bodies and neurites in the cerebral cortex correlate with the presence of dementia in Parkinson disease (PD) and Dementia with Lewy bodies (DLB), but whether αS influences synaptic vesicle dynamics in human cortical neurons is unknown. Using a new iPSC-based assay platform for measuring synaptic vesicle cycling, we found that in human cortical glutamatergic neurons, increased αS from either transgenic expression or triplication of the endogenous locus in patient-derived neurons reduced synaptic vesicle cycling under both stimulated and spontaneous conditions. Thus, using a robust, easily adopted assay platform, we show for the first time αS-induced synaptic dysfunction in human cortical neurons, a key cellular substrate for PD dementia and DLB.
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The neuronal protein α-synuclein is centrally involved in the neurodegeneration occurring in Parkinson's disease and related synucleinopathies. α-Synuclein's membrane-induced 3-11 helix conformation has a hydrophobic membrane-embedded half and a hydrophilic cytosolic half. Here, we studied the significance of (a) the surprising hydrophobicity of amino-acids at cytosol-exposed helix position 8; (b) the absence of positively charged lysine/arginine from all cytosol-exposed positions (1-5-8-9). We found that (a) further increasing hydrophobicity or adding lysine, but not glutamate, at position 8 augments both membrane interaction and S129 phosphorylation; (b) adding lysines at cytosol-exposed positions 1, 5, 8, or 9 has similar effects. Variants abundantly present in membranes by biochemical fractionation markedly colocalized with transferrin-receptor (an endosomal marker) in immunofluorescence-microscopy, indicating accumulation at vesicle membranes. Thus, we observed a striking correlation between membrane attraction and S129 phosphorylation, relevant for understanding α-synuclein biology in health and disease.
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Lisina , alfa-Sinucleína , alfa-Sinucleína/metabolismo , Fosforilação , Citosol/metabolismo , Lisina/metabolismo , Interações Hidrofóbicas e HidrofílicasRESUMO
α-Synuclein phosphorylation at serine-129 (pS129) is a widely used surrogate marker of pathology in Parkinson's disease and other synucleinopathies. However, we recently demonstrated that phosphorylation of S129 is also a physiological activator of synaptic transmission. In a feed-forward fashion, neuronal activity triggers reversible pS129. Here, we show that Parkinson's disease-linked missense mutations in SNCA impact activity-dependent pS129. Under basal conditions, cytosol-enriched A30P, H50Q, and G51D mutant forms of α-synuclein exhibit reduced pS129 levels in rat primary cortical neurons. A53T pS129 levels are similar to wild-type, and E46K pS129 levels are higher. A30P and E46K mutants show impaired reversibility of pS129 after stimulation. For the engineered profoundly membrane-associated α-synuclein mutant "3K" (E35K + E46K + E61K), de-phosphorylation was virtually absent after blocking stimulation, implying that reversible pS129 is severely compromised. Importantly, pS129 excess resulting from proteasome inhibition is also associated with reduced reversibility by neuronal inhibition, kinase inhibition, or phosphatase activation. Our findings suggest that perturbed pS129 dynamics are probably a shared characteristic of pathology-associated α-synuclein, with possible implications for synucleinopathy treatment and diagnosis.
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Doença de Parkinson , Sinucleinopatias , Animais , Ratos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Serina/metabolismo , FosforilaçãoRESUMO
We previously reported that prolonged exposure to an enriched environment (EE) enhances hippocampal synaptic plasticity, with one of the significant mechanistic pathways being activation of ß2-adrenergic receptor (ß2-AR) signaling, thereby mitigating the synaptotoxic effects of soluble oligomers of amyloid ß-protein (oAß). However, the detailed mechanism remained elusive. In this work, we recorded field excitatory postsynaptic potentials (fEPSP) in the CA1 region of mouse hippocampal slices treated with or without toxic Aß-species. We found that pharmacological activation of ß2-AR, but not ß1-AR, selectively mimicked the effects of EE in enhancing LTP and preventing oAß-induced synaptic dysfunction. Mechanistic analyses showed that certain histone deacetylase (HDAC) inhibitors mimicked the benefits of EE, but this was not seen in ß2-AR knockout mice, suggesting that activating ß2-AR prevents oAß-mediated synaptic dysfunction via changes in histone acetylation. EE or activation of ß-ARs each decreased HDAC2, whereas Aß oligomers increased HDAC2 levels in the hippocampus. Further, oAß-induced inflammatory effects and neurite degeneration were prevented by either ß2-AR agonists or certain specific HDAC inhibitors. These preclinical results suggest that activation of ß2-AR is a novel potential therapeutic strategy to mitigate oAß-mediated features of AD.
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Peptídeos beta-Amiloides , Hipocampo , Camundongos , Animais , Peptídeos beta-Amiloides/toxicidade , Peptídeos beta-Amiloides/metabolismo , Transdução de Sinais , Epigênese Genética , Camundongos KnockoutRESUMO
In Parkinson's disease and other synucleinopathies, the elevation of α-synuclein phosphorylated at Serine129 (pS129) is a widely cited marker of pathology. However, the physiological role for pS129 has remained undefined. Here we use multiple approaches to show for the first time that pS129 functions as a physiological regulator of neuronal activity. Neuronal activity triggers a sustained increase of pS129 in cultured neurons (200% within 4 h). In accord, brain pS129 is elevated in environmentally enriched mice exhibiting enhanced long-term potentiation. Activity-dependent α-synuclein phosphorylation is S129-specific, reversible, confers no cytotoxicity, and accumulates at synapsin-containing presynaptic boutons. Mechanistically, our findings are consistent with a model in which neuronal stimulation enhances Plk2 kinase activity via a calcium/calcineurin pathway to counteract PP2A phosphatase activity for efficient phosphorylation of membrane-bound α-synuclein. Patch clamping of rat SNCA-/- neurons expressing exogenous wild-type or phospho-incompetent (S129A) α-synuclein suggests that pS129 fine-tunes the balance between excitatory and inhibitory neuronal currents. Consistently, our novel S129A knock-in (S129AKI) mice exhibit impaired hippocampal plasticity. The discovery of a key physiological function for pS129 has implications for understanding the role of α-synuclein in neurotransmission and adds nuance to the interpretation of pS129 as a synucleinopathy biomarker.
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Alpha-synuclein (αS), the key protein in Parkinson's disease, is typically described as an intrinsically disordered protein. Consistent with this notion, several context-dependent folding states may coexist in neurons. Unfolded soluble monomers, helical monomers at membranes and helical multimers (soluble or at membranes) have all been reported and may be in an equilibrium with each other. We previously found that αS can be stabilized in its membrane-associated monomeric form by genetically increasing the hydrophobicity of the membrane-embedded half of the αS helix. αS amphipathic helix formation at membranes is governed by up to nine 11-amino acid repeats with the core motif KTKEGV. However, this repeat is only imperfectly conserved; for example, it consists of KAKEGV in repeat #1, KTKEQV in repeat #5, and AVVTGV in the poorly conserved repeat #6. Here we explored the effect of perfecting the αS core repeat to nine times KTKEGV ("9KV") and found by sequential protein extraction that this engineered mutant accumulates in the cytosolic phase of neural cells. Intact-cell cross-linking trapped a part of the cytosolic portion at multimeric positions (30, 60, 80, 100 kDa). Thus, compared to wild-type αS, αS 9KV seems less prone to populating the membrane-associated monomeric form. Removing the "ATVA" intervening amino-acid sequence between repeats 4 and 5 slightly increased cytosolic localization while adding "ATVA" in between all repeats 1-8 caused αS to be trapped as a monomer in membrane fractions. Our results contribute to an ongoing debate on the dynamic structure of αS, highlighting that wild-type αS is unlikely to be fully multimeric/monomeric or fully cytosolic/membrane-associated in cells, but protein engineering can create αS variants that preferentially adopt a certain state. Overall, the imperfect nature of the KTKEGV repeat motifs and the presence of ATVA in between repeats 4 and 5 seem to prevent a strong cytosolic localization of αS and thus play a major role in the protein's ability to dynamically populate cytosolic vs. membrane-associated and monomeric vs. multimeric states.
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Doença de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , Solubilidade , Mutação , Doença de Parkinson/metabolismo , Sequência de AminoácidosRESUMO
α-Synuclein (αS) plays a key role in Parkinson's disease. Although Parkinson's disease is typically "sporadic," inherited αS missense mutations provide crucial insights into molecular mechanisms. Here, we examine two clinical mutants, E46K and G51D, which are both in the conserved N-terminus that mediates transient αS-membrane interactions. However, E46K increases and G51D decreases αS-membrane interactions. Previously, we amplified E46K via the 11-residue repeat motifs, creating "3K" (E35K+E46K+E61K). Here, we engineered these motifs to amplify G51D (V40D+G51D+V66D = "3D") and systematically compared E46K/3K versus G51D/3D. We found that G51D increased cytosolic αS in neural cells and 3D aggravates this. G51D, and 3D even more, reduced αS multimer-to-monomer (αS60:αS14) ratio. Both amplified variants caused cellular stress in rat primary neurons and reduced growth in human neuroblastoma cells. Importantly, both 3K- and 3D-induced stress was ameliorated by pharmacologically inhibiting stearoyl-CoA desaturase or by conditioning the cells in palmitic (16:0) or myristic (14:0) acid. SCD inhibition lowered lipid-droplet accumulation in both 3D- and 3K-expressing cells and benefitted G51D by normalizing multimer:monomer ratio, as reported previously for E46K. Our findings suggest that, despite divergent cytosol/membrane partitioning, both G51D and E46K neurotoxicity can be prevented by decreasing fatty-acid unsaturation as a common therapeutic approach.SIGNIFICANCE STATEMENT α-Synuclein (αS) dyshomeostasis is linked to Parkinson's disease. Here we focus on two contrasting familial-Parkinson's disease αS mutants, E46K and G51D, that alter αS membrane association in opposite directions (E46K increases, G51D decreases it). Taking advantage of αS repeat structure, here we designed αS "3D," an amplified G51D variant (V40D+G51D+V66D). αS 3D further enhanced G51D's cytosolic enrichment. Systematic comparison of G51D/3D with membrane-enriched E46K/its amplified variant 3K revealed that both can elicit stress in human neural cells and primary rodent neurons. This toxicity can be ameliorated by inhibiting stearoyl-CoA desaturase or by saturated fatty acid conditioning. Thus, despite divergent membrane binding, both G51D and E46K αS dyshomeostasis are mitigated by altering fatty acid saturation as a shared target.
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Ácidos Graxos , Doença de Parkinson , alfa-Sinucleína , Animais , Citosol/metabolismo , Ácidos Graxos/metabolismo , Homeostase , Doença de Parkinson/metabolismo , Ratos , Estearoil-CoA Dessaturase/metabolismo , alfa-Sinucleína/metabolismoRESUMO
α-Synuclein (αS) has been well-documented to play a role in human synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). First, the lesions found in PD/DLB brains-Lewy bodies and Lewy neurites-are rich in aggregated αS. Second, genetic evidence links missense mutations and increased αS expression to familial forms of PD/DLB. Third, toxicity and cellular stress can be caused by αS under certain experimental conditions. In contrast, the homologs ß-synuclein (ßS) and γ-synuclein (γS) are not typically found in Lewy bodies/neurites, have not been clearly linked to brain diseases and have been largely non-toxic in experimental settings. In αS, the so-called non-amyloid-ß component of plaques (NAC) domain, constituting amino acids 61-95, has been identified to be critical for aggregation in vitro. This domain is partially absent in ßS and only incompletely conserved in γS, which could explain why both homologs do not cause disease. However, αS in vitro aggregation and cellular toxicity have not been firmly linked experimentally, and it has been proposed that excess αS membrane binding is sufficient to induce neurotoxicity. Indeed, recent characterizations of Lewy bodies have highlighted the accumulation of lipids and membranous organelles, raising the possibility that ßS and γS could also become neurotoxic if they were more prone to membrane/lipid binding. Here, we increased ßS and γS membrane affinity by strategic point mutations and demonstrate that these proteins behave like membrane-associated monomers, are cytotoxic and form round cytoplasmic inclusions that can be prevented by inhibiting stearoyl-CoA desaturase.
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Membrana Celular/metabolismo , Corpos de Inclusão/metabolismo , alfa-Sinucleína/metabolismo , beta-Sinucleína/metabolismo , gama-Sinucleína/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Humanos , Mutagênese , Agregação Patológica de Proteínas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Solubilidade , alfa-Sinucleína/química , alfa-Sinucleína/genética , beta-Sinucleína/química , beta-Sinucleína/genética , gama-Sinucleína/química , gama-Sinucleína/genéticaRESUMO
Aggregation of α-synuclein (αS) leads to the hallmark neuropathology of Parkinson's disease (PD) and related synucleinopathies. αS has been described to exist in both cytosolic and membrane-associated forms, the relative abundance of which has remained unsettled. To study αS under the most relevant conditions by a quantitative method, we cultured and matured rodent primary cortical neurons for >17 days and determined αS cytosol:membrane distribution via centrifugation-free sequential extractions based on the weak ionic detergent digitonin. We noticed that at lower temperatures (4 °C or room temperature), αS was largely membrane-associated. At 37 °C, however, αS solubility was markedly increased. In contrast, the extraction of control proteins (GAPDH, cytosolic; calnexin, membrane) was not affected by temperature. When we compared the relative distribution of the synuclein homologs αS and ß-synuclein (ßS) under various conditions that differed in temperature and digitonin concentration (200-1200 µg/ml), we consistently found αS to be more membrane-associated than ßS. Both proteins, however, exhibited temperature-dependent membrane binding. Under the most relevant conditions (37 °C and 800 µg/ml digitonin, i.e., the lowest digitonin concentration that extracted cytosolic GAPDH to near completion), cytosolic distribution was 49.8% ± 9.0% for αS and 63.6% ± 6.6% for ßS. PD-linked αS A30P was found to be largely cytosolic, confirming previous studies that had used different methods. Our work highlights the dynamic nature of cellular synuclein behavior and has important implications for protein-biochemical and cell-biological studies of αS proteostasis, such as testing the effects of genetic and pharmacological manipulations.
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Membrana Celular/genética , Neurônios/metabolismo , Doença de Parkinson/genética , alfa-Sinucleína/genética , beta-Sinucleína/genética , Sequência de Aminoácidos/genética , Animais , Membrana Celular/química , Humanos , Lentivirus/genética , Neurônios/química , Doença de Parkinson/imunologia , Doença de Parkinson/patologia , Cultura Primária de Células , Agregados Proteicos/genética , Agregados Proteicos/imunologia , Agregação Patológica de Proteínas/genética , Ligação Proteica/genética , Ratos , Temperatura , alfa-Sinucleína/química , alfa-Sinucleína/imunologia , alfa-Sinucleína/isolamento & purificação , beta-Sinucleína/química , beta-Sinucleína/imunologia , beta-Sinucleína/isolamento & purificaçãoRESUMO
BACKGROUND: Synucleinopathies, including Parkinson's disease (PD), are characterized by α-synuclein (αS) cytoplasmic inclusions. αS-dependent vesicle-trafficking defects are important in PD pathogenesis, but their mechanisms are not well understood. Protein palmitoylation, post-translational addition of the fatty acid palmitate to cysteines, promotes trafficking by anchoring specific proteins to the vesicle membrane. αS itself cannot be palmitoylated as it lacks cysteines, but it binds to membranes, where palmitoylation occurs, via an amphipathic helix. We hypothesized that abnormal αS membrane-binding impairs trafficking by disrupting palmitoylation. Accordingly, we investigated the therapeutic potential of increasing cellular palmitoylation. OBJECTIVES: We asked whether upregulating palmitoylation by inhibiting the depalmitoylase acyl-protein-thioesterase-1 (APT1) ameliorates pathologic αS-mediated cellular phenotypes and sought to identify the mechanism. METHODS: Using human neuroblastoma cells, rat neurons, and iPSC-derived PD patient neurons, we examined the effects of pharmacologic and genetic downregulation of APT1 on αS-associated phenotypes. RESULTS: APT1 inhibition or knockdown decreased αS cytoplasmic inclusions, reduced αS serine-129 phosphorylation (a PD neuropathological marker), and protected against αS-dependent neurotoxicity. We identified the APT1 substrate microtubule-associated-protein-6 (MAP6), which binds to vesicles in a palmitoylation-dependent manner, as a key mediator of these effects. Mechanistically, we found that pathologic αS accelerated palmitate turnover on MAP6, suggesting that APT1 inhibition corrects a pathological αS-dependent palmitoylation deficit. We confirmed the disease relevance of this mechanism by demonstrating decreased MAP6 palmitoylation in neurons from αS gene triplication patients. CONCLUSIONS: Our findings demonstrate a novel link between the fundamental process of palmitoylation and αS pathophysiology. Upregulating palmitoylation represents an unexplored therapeutic strategy for synucleinopathies. © 2020 International Parkinson and Movement Disorder Society.
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Doença de Parkinson , alfa-Sinucleína , Animais , Humanos , Lipoilação , Neurônios/metabolismo , Ratos , Regulação para Cima , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Genetic and biochemical evidence attributes neuronal loss in Parkinson's disease (PD) and related brain diseases to dyshomeostasis of the 14 kDa protein α-synuclein (αS). There is no consensus on how αS exerts toxicity. Explanations range from disturbed vesicle biology to proteotoxicity caused by fibrillar aggregates. To probe these mechanisms further, robust cellular toxicity models are needed, but their availability is limited. We previously reported that a shift from dynamic multimers to monomers is an early event in αS dyshomeostasis, as caused by familial PD (fPD)-linked mutants such as E46K. Excess monomers accumulate in round, lipid-rich inclusions. Engineered αS '3K' (E35K+E46K+E61K) amplifies E46K, causing a PD-like, L-DOPA-responsive motor phenotype in transgenic mice. Here, we present a cellular model of αS neurotoxicity after transducing human neuroblastoma cells to express yellow fluorescent protein (YFP)-tagged αS 3K in a doxycycline-dependent manner. αS-3K::YFP induction causes pronounced growth defects that accord with cell death. We tested candidate compounds for their ability to restore growth, and stearoyl-CoA desaturase (SCD) inhibitors emerged as a molecule class with growth-restoring capacity, but the therapeutic window varied among compounds. The SCD inhibitor MF-438 fully restored growth while exerting no apparent cytotoxicity. Our αS bioassay will be useful for elucidating compound mechanisms, for pharmacokinetic studies, and for compound/genetic screens.
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Proliferação de Células/efeitos dos fármacos , Neuroblastoma/metabolismo , Piridazinas/farmacologia , Estearoil-CoA Dessaturase/antagonistas & inibidores , Tiadiazóis/farmacologia , alfa-Sinucleína/genética , alfa-Sinucleína/toxicidade , Proteínas de Bactérias , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Doença por Corpos de Lewy/tratamento farmacológico , Doença por Corpos de Lewy/metabolismo , Proteínas Luminescentes , Mutação , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Estearoil-CoA Dessaturase/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Microscopy of Lewy bodies in Parkinson's disease (PD) suggests they are not solely filamentous deposits of α-synuclein (αS) but also contain vesicles and other membranous material. We previously reported the existence of native αS tetramers/multimers and described engineered mutations of the αS KTKEGV repeat motifs that abrogate the multimers. The resultant excess monomers accumulate in lipid membrane-rich inclusions associated with neurotoxicity exceeding that of natural familial PD mutants, such as E46K. Here, we use the αS "3K" (E35K+E46K+E61K) engineered mutation to probe the mechanisms of reported small-molecule modifiers of αS biochemistry and then identify compounds via a medium-throughput automated screen. αS 3K, which forms round, vesicle-rich inclusions in cultured neurons and causes a PD-like, l-DOPA-responsive motor phenotype in transgenic mice, was fused to YFP, and fluorescent inclusions were quantified. Live-cell microscopy revealed the highly dynamic nature of the αS inclusions: for example, their rapid clearance by certain known modulators of αS toxicity, including tacrolimus (FK506), isradipine, nilotinib, nortriptyline, and trifluoperazine. Our automated 3K cellular screen identified inhibitors of stearoyl-CoA desaturase (SCD) that robustly prevent the αS inclusions, reduce αS 3K neurotoxicity, and prevent abnormal phosphorylation and insolubility of αS E46K. SCD inhibition restores the E46K αS multimer:monomer ratio in human neurons, and it actually increases this ratio for overexpressed wild-type αS. In accord, conditioning 3K cells in saturated fatty acids rescued, whereas unsaturated fatty acids worsened, the αS phenotypes. Our cellular screen allows probing the mechanisms of synucleinopathy and refining drug candidates, including SCD inhibitors and other lipid modulators.
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Corpos de Inclusão/efeitos dos fármacos , Lipídeos/análise , Mutação , Neuroblastoma/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Estearoil-CoA Dessaturase/antagonistas & inibidores , alfa-Sinucleína/química , Animais , Linhagem Celular , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Estearoil-CoA Dessaturase/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
The contractile actin cortex is a thin layer of filamentous actin, myosin motors, and regulatory proteins beneath the plasma membrane crucial to cytokinesis, morphogenesis, and cell migration. However, the factors regulating actin assembly in this compartment are not well understood. Using the Dictyostelium model system, we show that the three Diaphanous-related formins (DRFs) ForA, ForE, and ForH are regulated by the RhoA-like GTPase RacE and synergize in the assembly of filaments in the actin cortex. Single or double formin-null mutants displayed only moderate defects in cortex function whereas the concurrent elimination of all three formins or of RacE caused massive defects in cortical rigidity and architecture as assessed by aspiration assays and electron microscopy. Consistently, the triple formin and RacE mutants encompassed large peripheral patches devoid of cortical F-actin and exhibited severe defects in cytokinesis and multicellular development. Unexpectedly, many forA- /E-/H- and racE- mutants protruded efficiently, formed multiple exaggerated fronts, and migrated with morphologies reminiscent of rapidly moving fish keratocytes. In 2D-confinement, however, these mutants failed to properly polarize and recruit myosin II to the cell rear essential for migration. Cells arrested in these conditions displayed dramatically amplified flow of cortical actin filaments, as revealed by total internal reflection fluorescence (TIRF) imaging and iterative particle image velocimetry (PIV). Consistently, individual and combined, CRISPR/Cas9-mediated disruption of genes encoding mDia1 and -3 formins in B16-F1 mouse melanoma cells revealed enhanced frequency of cells displaying multiple fronts, again accompanied by defects in cell polarization and migration. These results suggest evolutionarily conserved functions for formin-mediated actin assembly in actin cortex mechanics.
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Citoesqueleto de Actina/genética , Proteínas de Transporte/genética , Proteínas Contráteis/genética , Melanoma Experimental/genética , Citoesqueleto de Actina/química , Actinas/genética , Animais , Sistemas CRISPR-Cas , Movimento Celular/genética , Polaridade Celular/genética , Proteínas Contráteis/química , Dictyostelium/genética , Modelos Animais de Doenças , Forminas , Humanos , Melanoma Experimental/patologia , Camundongos , Microscopia Eletrônica , Contração Muscular/genética , Proteína rhoA de Ligação ao GTP/química , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
ß-Sheet-rich aggregates of α-synuclein (αS) are the hallmark neuropathology of Parkinson's disease (PD) and related synucleinopathies, whereas the native conformations of αS in healthy cells are under debate. Cross-linking analyses in intact cells detect a large portion of endogenous αS in apparent multimeric states, most notably as putative tetramers (αS60) that run around 60 kDa on SDS-PAGE, but also point at the dynamic nature of cellular αS states. Standardization of αS cross-linking methods will facilitate efforts to study the effects of genetic, pharmacological, and environmental factors on αS conformation. Here, we present detailed protocols for cross-linking cellular αS multimers in cultured cells and brain tissues. These protocols will benefit future studies aimed at characterizing αS conformation in its cellular environment, both at steady state and upon perturbation, be it chronic or acute.
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Modelos Moleculares , Conformação Proteica , alfa-Sinucleína/química , Células Cultivadas , Mutação , Doença de Parkinson/metabolismo , Multimerização Proteica , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
In Parkinson's disease (PD), α-synuclein (αS) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in αS or lipid/fatty acid homeostasis affect each other. Lipidomic profiling of human αS-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of αS dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased αS yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in αS-overexpressing rat neurons. In a C. elegans model, SCD knockout prevented αS-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on αS homeostasis: in human neural cells, excess OA caused αS inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for αS-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.
Assuntos
Antiparkinsonianos/farmacologia , Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolômica/métodos , Neurônios/efeitos dos fármacos , Doença de Parkinson/tratamento farmacológico , Estearoil-CoA Dessaturase/antagonistas & inibidores , alfa-Sinucleína/toxicidade , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Linhagem Celular , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Córtex Cerebral/patologia , Diglicerídeos/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/enzimologia , Neurônios Dopaminérgicos/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/enzimologia , Células-Tronco Pluripotentes Induzidas/patologia , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/enzimologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Terapia de Alvo Molecular , Degeneração Neural , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/enzimologia , Células-Tronco Neurais/patologia , Neurônios/enzimologia , Neurônios/patologia , Ácido Oleico/metabolismo , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Ratos Sprague-Dawley , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Estearoil-CoA Dessaturase/metabolismo , Triglicerídeos/metabolismo , alfa-Sinucleína/genéticaRESUMO
Reduced function of parkin appears to be a central pathogenic event in Parkinson disease (PD). Increasing parkin levels enhances survival in models of PD-related neuronal death and is a promising therapeutic objective. Previously, we demonstrated that the transcription factor ATF4 promotes survival in response to PD-mimetic stressors by maintaining parkin levels. ATF4 translation is up-regulated by phosphorylation of the translation initiation factor eIF2α. The small molecule guanabenz enhances eIF2α phosphorylation by blocking the function of GADD34, a regulatory protein that promotes eIF2α dephosphorylation. We tested the hypothesis that guanabenz, by inhibiting GADD34 and consequently increasing eIF2α phosphorylation and elevating ATF4, would improve survival in models of PD by up-regulating parkin. We found that GADD34 is strongly induced by 6-OHDA, and that GADD34 localization is dramatically altered in dopaminergic substantia nigra neurons in PD cases. We further demonstrated that guanabenz attenuates 6-hydroxydopamine (6-OHDA) induced cell death of differentiated PC12 cells and primary ventral midbrain dopaminergic neurons in culture, and of dopaminergic neurons in the substantia nigra of mice. In culture models, guanabenz also increases eIF2α phosphorylation and ATF4 and parkin levels in response to 6-OHDA. Furthermore, if either ATF4 or parkin is silenced, then the protective effect of guanabenz is lost. We also found similar results in a distinct model of neuronal death: primary cultures of cortical neurons treated with the topoisomerase I inhibitor camptothecin, in which guanabenz limited camptothecin-induced neuronal death in an ATF4- and parkin-dependent manner. In summary, our data suggest that guanabenz and other GADD34 inhibitors could be used as therapeutic agents to boost parkin levels and thereby slow neurodegeneration in PD and other neurodegenerative conditions.