RESUMEN
Alpha-synuclein (αS) is a conformationally plastic protein that reversibly binds to cellular membranes. It aggregates and is genetically linked to Parkinson's disease (PD). Here, we show that αS directly modulates processing bodies (P-bodies), membraneless organelles that function in mRNA turnover and storage. The N terminus of αS, but not other synucleins, dictates mutually exclusive binding either to cellular membranes or to P-bodies in the cytosol. αS associates with multiple decapping proteins in close proximity on the Edc4 scaffold. As αS pathologically accumulates, aberrant interaction with Edc4 occurs at the expense of physiologic decapping-module interactions. mRNA decay kinetics within PD-relevant pathways are correspondingly disrupted in PD patient neurons and brain. Genetic modulation of P-body components alters αS toxicity, and human genetic analysis lends support to the disease-relevance of these interactions. Beyond revealing an unexpected aspect of αS function and pathology, our data highlight the versatility of conformationally plastic proteins with high intrinsic disorder.
Asunto(s)
Enfermedad de Parkinson , alfa-Sinucleína , Humanos , Enfermedad de Parkinson/metabolismo , Cuerpos de Procesamiento , Estabilidad del ARN , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Calcineurin is an essential Ca2+-dependent phosphatase. Increased calcineurin activity is associated with α-synuclein (α-syn) toxicity, a protein implicated in Parkinson's Disease (PD) and other neurodegenerative diseases. Calcineurin can be inhibited with Tacrolimus through the recruitment and inhibition of the 12-kDa cis-trans proline isomerase FK506-binding protein (FKBP12). Whether calcineurin/FKBP12 represents a native physiologically relevant assembly that occurs in the absence of pharmacological perturbation has remained elusive. We leveraged α-syn as a model to interrogate whether FKBP12 plays a role in regulating calcineurin activity in the absence of Tacrolimus. We show that FKBP12 profoundly affects the calcineurin-dependent phosphoproteome, promoting the dephosphorylation of a subset of proteins that contributes to α-syn toxicity. Using a rat model of PD, partial elimination of the functional interaction between FKBP12 and calcineurin, with low doses of the Food and Drug Administration (FDA)-approved compound Tacrolimus, blocks calcineurin's activity toward those proteins and protects against the toxic hallmarks of α-syn pathology. Thus, FKBP12 can endogenously regulate calcineurin activity with therapeutic implications for the treatment of PD.
Asunto(s)
Calcineurina/metabolismo , Enfermedad de Parkinson/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteína 1A de Unión a Tacrolimus/metabolismo , alfa-Sinucleína/metabolismo , Animales , Calcineurina/genética , Modelos Animales de Enfermedad , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Fosfoproteínas/genética , Proteoma/genética , Ratas , Ratas Sprague-Dawley , Tacrolimus/farmacología , Proteína 1A de Unión a Tacrolimus/genética , alfa-Sinucleína/genéticaRESUMEN
Calcineurin (CN) is a highly conserved Ca(2+)-calmodulin (CaM)-dependent phosphatase that senses Ca(2+) concentrations and transduces that information into cellular responses. Ca(2+) homeostasis is disrupted by α-synuclein (α-syn), a small lipid binding protein whose misfolding and accumulation is a pathological hallmark of several neurodegenerative diseases. We report that α-syn, from yeast to neurons, leads to sustained highly elevated levels of cytoplasmic Ca(2+), thereby activating a CaM-CN cascade that engages substrates that result in toxicity. Surprisingly, complete inhibition of CN also results in toxicity. Limiting the availability of CaM shifts CN's spectrum of substrates toward protective pathways. Modulating CN or CN's substrates with highly selective genetic and pharmacological tools (FK506) does the same. FK506 crosses the blood brain barrier, is well tolerated in humans, and is active in neurons and glia. Thus, a tunable response to CN, which has been conserved for a billion years, can be targeted to rebalance the phosphatase's activities from toxic toward beneficial substrates. These findings have immediate therapeutic implications for synucleinopathies.
Asunto(s)
Calcineurina/metabolismo , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidad , Animales , Calcineurina/genética , Inhibidores de la Calcineurina , Señalización del Calcio , Calmodulina/metabolismo , Células Cultivadas , Técnicas de Silenciamiento del Gen , Humanos , Enfermedad por Cuerpos de Lewy/metabolismo , Ratones , Ratones Transgénicos , Modelos Neurológicos , Factores de Transcripción NFATC/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Enfermedad de Parkinson/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidad , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/toxicidad , Tacrolimus/farmacología , alfa-Sinucleína/genéticaRESUMEN
No disease-modifying therapies are available for synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple systems atrophy (MSA). The lack of therapies has been impeded by a paucity of validated drug targets and problematic cell-based model systems. New approaches are therefore needed to identify genes and compounds that directly target the underlying cellular pathologies elicited by the pathological protein, α-synuclein (α-syn). This small, lipid-binding protein impinges on evolutionarily conserved processes such as vesicle trafficking and mitochondrial function. For decades, the genetically tractable, single-cell eukaryote, budding yeast, has been used to study nearly all aspects of cell biology. More recently, yeast has revealed key insights into the underlying cellular pathologies caused by α-syn. The robust cellular toxicity caused by α-syn expression facilitates unbiased high-throughput small-molecule screening. Critically, one must validate the discoveries made in yeast in disease-relevant neuronal models. Here, we describe two recent reports that together establish yeast-to-human discovery platforms for synucleinopathies. In this exemplar, genes and small molecules identified in yeast were validated in patient-derived neurons that present the same cellular phenotypes initially discovered in yeast. On validation, we returned to yeast, where unparalleled genetic approaches facilitated the elucidation of a small molecule's mode of action. This approach enabled the identification and neuronal validation of a previously unknown "druggable" node that interfaces with the underlying, precipitating pathologies caused by α-syn. Such platforms can provide sorely needed leads and fresh ideas for disease-modifying therapy for these devastating diseases.
Asunto(s)
Trastornos del Movimiento/patología , Neuronas/metabolismo , Investigación Biomédica Traslacional , Levaduras , alfa-Sinucleína/metabolismo , Animales , Humanos , Trastornos del Movimiento/genética , Levaduras/genética , Levaduras/metabolismo , alfa-Sinucleína/genéticaRESUMEN
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.
RESUMEN
α-Synuclein is a key molecule in the pathogenesis of synucleinopathy including dementia with Lewy bodies, Parkinson's disease, and multiple system atrophy. Sirtuins are NAD(+)-dependent protein deacetylases that are highly conserved and counter aging in lower organisms. We show that the life span of a mouse model with A53T α-synuclein mutation is increased by overexpressing SIRT1 and decreased by knocking out SIRT1 in brain. Furthermore, α-synuclein aggregates are reduced in the brains of mice with SIRT1 overexpression and increased by SIRT1 deletion. We show that SIRT1 deacetylates HSF1 (heat shock factor 1) and increases HSP70 RNA and protein levels, but only in the brains of mice with A53T and SIRT1 expression. Thus, SIRT1 responds to α-synuclein aggregation-induced stress by activating molecular chaperones to protect against disease.
Asunto(s)
Encéfalo/metabolismo , Cuerpos de Inclusión/metabolismo , Chaperonas Moleculares/fisiología , Sirtuina 1/genética , Estrés Fisiológico/fisiología , alfa-Sinucleína/metabolismo , Animales , Modelos Animales de Enfermedad , Cuerpos de Inclusión/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Sirtuina 1/deficiencia , alfa-Sinucleína/genéticaRESUMEN
Deregulated de novo lipid synthesis (DNLS) is a potential druggable vulnerability in glioblastoma (GBM), a highly lethal and incurable cancer. Yet the molecular mechanisms that determine susceptibility to DNLS-targeted therapies remain unknown, and the lack of brain-penetrant inhibitors of DNLS has prevented their clinical evaluation as GBM therapeutics. Here, we report that YTX-7739, a clinical-stage inhibitor of stearoyl CoA desaturase (SCD), triggers lipotoxicity in patient-derived GBM stem-like cells (GSCs) and inhibits fatty acid desaturation in GSCs orthotopically implanted in mice. When administered as a single agent, or in combination with temozolomide (TMZ), YTX-7739 showed therapeutic efficacy in orthotopic GSC mouse models owing to its lipotoxicity and ability to impair DNA damage repair. Leveraging genetic, pharmacological, and physiological manipulation of key signaling nodes in gliomagenesis complemented with shotgun lipidomics, we show that aberrant MEK/ERK signaling and its repression of the energy sensor AMP-activated protein kinase (AMPK) primarily drive therapeutic vulnerability to SCD and other DNLS inhibitors. Conversely, AMPK activation mitigates lipotoxicity and renders GSCs resistant to the loss of DNLS, both in culture and in vivo, by decreasing the saturation state of phospholipids and diverting toxic lipids into lipid droplets. Together, our findings reveal mechanisms of metabolic plasticity in GSCs and provide a framework for the rational integration of DNLS-targeted GBM therapies.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Animales , Ratones , Glioblastoma/genética , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Quinasas Activadas por AMP/uso terapéutico , Línea Celular Tumoral , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Daño del ADN , Lípidos , Células Madre Neoplásicas/metabolismoRESUMEN
The development of phenotypic assays with appropriate analyses is an important step in the drug discovery process. Assays using induced pluripotent stem cell (iPSC)-derived human neurons are emerging as powerful tools for drug discovery in neurological disease. We have previously shown that longitudinal single cell tracking enabled the quantification of survival and death of neurons after overexpression of α-synuclein with a familial Parkinson's disease mutation (A53T). The reliance of this method on manual counting, however, rendered the process labor intensive, time consuming and error prone. To overcome these hurdles, we have developed automated detection algorithms for neurons using the BioStation CT live imaging system and CL-Quant software. In the current study, we use these algorithms to successfully measure the risk of neuronal death caused by overexpression of α-synuclein (A53T) with similar accuracy and improved consistency as compared to manual counting. This novel method also provides additional key readouts of neuronal fitness including total neurite length and the number of neurite nodes projecting from the cell body. Finally, the algorithm reveals the neuroprotective effects of brain-derived neurotrophic factor (BDNF) treatment in neurons overexpressing α-synuclein (A53T). These data show that an automated algorithm improves the consistency and considerably shortens the analysis time of assessing neuronal health, making this method advantageous for small molecule screening for inhibitors of synucleinopathy and other neurodegenerative diseases.
Asunto(s)
Sinucleinopatías , alfa-Sinucleína , Humanos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Sinucleinopatías/metabolismo , Rastreo Celular , Neuronas/metabolismo , AlgoritmosRESUMEN
In Parkinson's disease (PD), dopaminergic (DA) neurons in the substantia nigra (SN, A9) are particularly vulnerable, compared to adjacent DA neurons within the ventral tegmental area (VTA, A10). Here, we show that in rat and human, one RAB3 isoform, RAB3B, has higher expression levels in A10 compared to A9 neurons. RAB3 is a monomeric GTPase protein that is highly enriched in synaptic vesicles and is involved in synaptic vesicle trafficking and synaptic transmission, disturbances of which have been implicated in several neurodegenerative diseases, including PD. These findings prompted us to further investigate the biology and neuroprotective capacity of RAB3B both in vitro and in vivo. RAB3B overexpression in human dopaminergic BE (2)-M17 cells increased neurotransmitter content, [(3)H] dopamine uptake, and levels of presynaptic proteins. AAV-mediated RAB3B overexpression in A9 DA neurons of the rat SN increased striatal dopamine content, number and size of synaptic vesicles, and levels of the presynaptic proteins, confirming in vitro findings. Measurement of extracellular DOPAC, a dopamine metabolite, following l-DOPA injection supported a role for RAB3B in enhancing the dopamine storage capacity of synaptic terminals. RAB3B overexpression in BE (2)-M17 cells was protective against toxins that simulate aspects of PD in vitro, including an oxidative stressor 6-hydroxydopamine (6-OHDA) and a proteasome inhibitor MG-132. Furthermore, RAB3B overexpression in rat SN both protected A9 DA neurons and resulted in behavioral improvement in a 6-OHDA retrograde lesion model of PD. These results suggest that RAB3B improves dopamine handling and storage capacity at presynaptic terminals, and confers protection to vulnerable DA neurons.
Asunto(s)
Dopamina/metabolismo , Terminales Presinápticos/metabolismo , Proteínas de Unión al GTP rab3/genética , Proteínas de Unión al GTP rab3/metabolismo , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Línea Celular , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Técnicas In Vitro , Leupeptinas/toxicidad , Levodopa/farmacología , Modelos Neurológicos , Oxidopamina/toxicidad , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/patología , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/ultraestructura , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Sustancia Negra/metabolismo , Vesículas Sinápticas/metabolismo , Área Tegmental Ventral/metabolismoRESUMEN
Stearoyl-CoA desaturase (SCD) is a potential therapeutic target for Parkinson's and related neurodegenerative diseases. SCD inhibition ameliorates neuronal toxicity caused by aberrant α-synuclein, a lipid-binding protein implicated in Parkinson's disease. Its inhibition depletes monounsaturated fatty acids, which may modulate α-synuclein conformations and membrane interactions. Herein, we characterize the pharmacokinetic and pharmacodynamic properties of YTX-7739, a clinical-stage SCD inhibitor. Administration of YTX-7739 to rats and monkeys for 15 days caused a dose-dependent increase in YTX-7739 concentrations that were well-tolerated and associated with concentration-dependent reductions in the fatty acid desaturation index (FADI), the ratio of monounsaturated to saturated fatty acids. An approximate 50% maximal reduction in the carbon-16 desaturation index was observed in the brain, with comparable responses in the plasma and skin. A study with a diet supplemented in SCD products indicates that changes in brain C16 desaturation were due to local SCD inhibition, rather than to changes in systemic fatty acids that reach the brain. Assessment of pharmacodynamic response onset and reversibility kinetics indicated that approximately 7 days of dosing were required to achieve maximal responses, which persisted for at least 2 days after cessation of dosing. YTX-7739 thus achieved sufficient concentrations in the brain to inhibit SCD and produce pharmacodynamic responses that were well-tolerated in rats and monkeys. These results provide a framework for evaluating YTX-7739 pharmacology clinically as a disease-modifying therapy to treat synucleinopathies.
Asunto(s)
Enfermedad de Parkinson , Estearoil-CoA Desaturasa , Animales , Ácidos Grasos/metabolismo , Ácidos Grasos/farmacología , Metabolismo de los Lípidos/fisiología , Ratas , Estearoil-CoA Desaturasa/metabolismo , alfa-Sinucleína/metabolismoRESUMEN
There are currently no preventive or disease-modifying therapies for Parkinson's Disease (PD). Failures in clinical trials necessitate a re-evaluation of existing pre-clinical models in order to adopt systems that better recapitulate underlying disease mechanisms and better predict clinical outcomes. In recent years, models utilizing patient-derived induced pluripotent stem cells (iPSC) have emerged as attractive models to recapitulate disease-relevant neuropathology in vitro without exogenous overexpression of disease-related pathologic proteins. Here, we utilized iPSC derived from patients with early-onset PD and dementia phenotypes that harbored either a point mutation (A53T) or multiplication at the α-synuclein/SNCA gene locus. We generated a three-dimensional (3D) cortical neurosphere culture model to better mimic the tissue microenvironment of the brain. We extensively characterized the differentiation process using quantitative PCR, Western immunoblotting and immunofluorescence staining. Differentiated and aged neurospheres revealed alterations in fatty acid profiles and elevated total and pathogenic phospho-α-synuclein levels in both A53T and the triplication lines compared to their isogenic control lines. Furthermore, treatment of the neurospheres with a small molecule inhibitor of stearoyl CoA desaturase (SCD) attenuated the protein accumulation and aberrant fatty acid profile phenotypes. Our findings suggest that the 3D cortical neurosphere model is a useful tool to interrogate targets for PD and amenable to test small molecule therapeutics.
Asunto(s)
Células Madre Pluripotentes Inducidas , Enfermedad de Parkinson , Humanos , alfa-Sinucleína/genética , Enfermedad de Parkinson/genética , Organoides , Ácidos GrasosRESUMEN
Increasing evidence has shown that Parkinson's disease (PD) impairs midbrain dopaminergic, cortical and other neuronal subtypes in large part due to the build-up of lipid- and vesicle-rich α-synuclein (αSyn) cytotoxic inclusions. We previously identified stearoyl-CoA desaturase (SCD) as a potential therapeutic target for synucleinopathies. A brain-penetrant SCD inhibitor, YTX-7739, was developed and has entered Phase 1 clinical trials. Here, we report the efficacy of YTX-7739 in reversing pathological αSyn phenotypes in various in vitro and in vivo PD models. In cell-based assays, YTX-7739 decreased αSyn-mediated neuronal death, reversed the abnormal membrane interaction of amplified E46K ("3K") αSyn, and prevented pathological phenotypes in A53T and αSyn triplication patient-derived neurospheres, including dysregulated fatty acid profiles and pS129 αSyn accumulation. In 3K PD-like mice, YTX-7739 crossed the blood-brain barrier, decreased unsaturated fatty acids, and prevented progressive motor deficits. Both YTX-7739 treatment and decreasing SCD activity through deletion of one copy of the SCD1 gene (SKO) restored the physiological αSyn tetramer-to-monomer ratio, dopaminergic integrity, and neuronal survival in 3K αSyn mice. YTX-7739 efficiently reduced pS129 + and PK-resistant αSyn in both human wild-type αSyn and 3K mutant mice similar to the level of 3K-SKO. Together, these data provide further validation of SCD as a PD therapeutic target and YTX-7739 as a clinical candidate for treating human α-synucleinopathies.
Asunto(s)
Enfermedad de Parkinson , alfa-Sinucleína , Animales , Encéfalo/metabolismo , Humanos , Ratones , Neuronas/metabolismo , Enfermedad de Parkinson/genética , Estearoil-CoA Desaturasa/genética , Estearoil-CoA Desaturasa/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
In Parkinson's disease (PD), loss of striatal dopaminergic (DA) terminals and degeneration of DA neurons in the substantia nigra (SN) are associated with glial reactions. Such inflammatory processes are commonly considered an epiphenomenon of neuronal degeneration. However, there is increasing recognition of the role of neuroinflammation as an initiation factor of DA neuron degeneration. To investigate this issue, we established a new model of brain inflammation by injecting the Toll-like receptor 3 (TLR-3) agonist polyinosinic:polycytidylic acid [poly(I:C)] in the SN of adult rats. Poly(I:C) injection induced a sustained inflammatory reaction in the SN and in the dorsolateral striatum. Significant changes were detected in proteins relevant to synaptic transmission and axonal transport. In addition, cytoplasmic mislocalization of neuronal TAR DNA binding protein TDP-43 was observed. Poly(I:C) injection increased the susceptibility of midbrain DA neurons to a subsequent neurotoxic trigger (low-dose 6-hydroxydopamine). Systemic delivery of interleukin-1 receptor antagonist protected SN DA neurons exposed to combined poly(I:C) induced inflammatory and neurotoxic oxidative stress. These data indicate that viral-like neuroinflammation induces predegenerative changes in the DA system, which lowers the set point toward neuronal dysfunction and degeneration. New powerful neuroprotective therapies for PD might be considered by targeting critical inflammatory mechanisms, including cytokine-induced neurotoxicity.
Asunto(s)
Cuerpo Estriado/patología , Dopamina , Degeneración Nerviosa/patología , Poli I-C/toxicidad , Sustancia Negra/patología , Receptor Toll-Like 3/agonistas , Animales , Cuerpo Estriado/efectos de los fármacos , Dopamina/fisiología , Femenino , Inyecciones Intraventriculares , Degeneración Nerviosa/inducido químicamente , Poli I-C/administración & dosificación , Ratas , Ratas Sprague-Dawley , Sustancia Negra/efectos de los fármacos , Receptor Toll-Like 3/fisiologíaRESUMEN
SLC18A2 encodes the vesicular monoamine transporter 2 protein that regulates neurotransmission and reduces cytosolic toxicity of monoamines. Deletion of this gene causes lethality in mice, and DNA sequence variation in this gene is associated with alcoholism and Parkinson's disease, among other disorders. The Caucasian SLC18A2 promoter has at least 20 haplotypes (A-T), with A representing two-thirds of 1460 chromosomes. It is not known why A is selected in the human lineage. To understand the selection, here we took a functional approach by investigating the regulations of 4 representative haplotypes (A, C, G, and T) by 17 agents. We show that 76.5% of the agents were able to regulate A but only 11.8-23.5% of them regulated the 3 other infrequent ones, observing a positive correlation between haplotype frequency and regulatability. Pathway and molecular analyses revealed five signaling hubs that regulate the four haplotypes differentially, probably through targeting the polymorphic core promoter region. These findings suggest that greater diversity of transcriptional regulations is the driving force for the haplotype selection in SLC18A2.
Asunto(s)
Regulación de la Expresión Génica , Selección Genética , Proteínas de Transporte Vesicular de Monoaminas/genética , Línea Celular , Variación Genética , Haplotipos , Humanos , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
Two adjacent groups of midbrain dopaminergic neurons, A9 (substantia nigra pars compacta) and A10 (ventral tegmental area), have distinct projections and exhibit differential vulnerability in Parkinson's disease. Little is known about transcription factors that influence midbrain dopaminergic subgroup phenotypes or their potential role in disease. Here, we demonstrate elevated expression of the transcription factor orthodenticle homeobox 2 in A10 dopaminergic neurons of embryonic and adult mouse, primate and human midbrain. Overexpression of orthodenticle homeobox 2 using lentivirus increased levels of known A10 elevated genes, including neuropilin 1, neuropilin 2, slit2 and adenylyl cyclase-activating peptide in both MN9D cells and ventral mesencephalic cultures, whereas knockdown of endogenous orthodenticle homeobox 2 levels via short hairpin RNA reduced expression of these genes in ventral mesencephalic cultures. Lack of orthodenticle homeobox 2 in the ventral mesencephalon of orthodenticle homeobox 2 conditional knockout mice caused a reduction of midbrain dopaminergic neurons and selective loss of A10 dopaminergic projections. Orthodenticle homeobox 2 overexpression protected dopaminergic neurons in ventral mesencephalic cultures from Parkinson's disease-relevant toxin, 1-methyl-4-phenylpyridinium, whereas downregulation of orthodenticle homeobox 2 using short hairpin RNA increased their susceptibility. These results show that orthodenticle homeobox 2 is important for establishing subgroup phenotypes of post-mitotic midbrain dopaminergic neurons and may alter neuronal vulnerability.
Asunto(s)
Axones/fisiología , Dopamina/fisiología , Mesencéfalo/fisiología , Neuronas/fisiología , Factores de Transcripción Otx/fisiología , 1-Metil-4-fenilpiridinio/toxicidad , Animales , Axones/efectos de los fármacos , Axones/patología , Células Cultivadas , Femenino , Humanos , Masculino , Mesencéfalo/efectos de los fármacos , Mesencéfalo/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mitosis , Neuronas/efectos de los fármacos , Neuronas/patología , FenotipoRESUMEN
Little is known about key pathological events preceding overt neuronal degeneration in Parkinson's disease (PD) and alpha-synucleinopathy. Recombinant adeno-associated virus 2-mediated delivery of mutant (A53T) human alpha-synuclein into the substantia nigra (SN) under a neuron-specific synapsin promoter resulted in protracted neurodegeneration with significant dopaminergic (DA) neuron loss by 17 weeks. As early as 4 weeks, there was an increase in a dopamine metabolite, DOPAC and histologically, DA axons in the striatum were dystrophic with degenerative bulbs. Before neuronal loss, significant changes were identified in levels of proteins relevant to synaptic transmission and axonal transport in the striatum and the SN. For example, striatal levels of rabphilin 3A and syntaxin were reduced. Levels of anterograde transport motor proteins (KIF1A, KIF1B, KIF2A, and KIF3A) were decreased in the striatum, whereas retrograde motor proteins (dynein, dynamitin, and dynactin1) were increased. In contrast to reduced levels in the striatum, KIF1A and KIF2A levels were elevated in the SN. There were dramatic changes in cytoskeletal protein levels, with actin levels increased and alpha-/gamma-tubulin levels reduced. In addition to these alterations, a neuroinflammatory response was observed at 8 weeks in the striatum, but not in the SN, demonstrated by increased levels of Iba-1, activated microglia and increased levels of proinflammatory cytokines, including IL-1beta, IFN-gamma and TNF-alpha. These results demonstrate that changes in proteins relevant to synaptic transmission and axonal transport coupled with neuroinflammation, precede alpha-synuclein-mediated neuronal death. These findings can provide ideas for antecedent biomarkers and presymptomatic interventions in PD.
Asunto(s)
Proteínas Portadoras/metabolismo , Dependovirus , Modelos Animales de Enfermedad , Dopamina/fisiología , Enfermedades del Sistema Nervioso/metabolismo , Terminales Presinápticos/metabolismo , alfa-Sinucleína/metabolismo , Animales , Transporte Axonal/genética , Proteínas Portadoras/genética , Muerte Celular/genética , Dependovirus/genética , Dopamina/genética , Femenino , Vectores Genéticos/genética , Humanos , Inflamación/metabolismo , Inflamación/patología , Inflamación/virología , Enfermedades del Sistema Nervioso/patología , Enfermedades del Sistema Nervioso/virología , Neuronas/metabolismo , Neuronas/patología , Neuronas/virología , Terminales Presinápticos/patología , Terminales Presinápticos/virología , Ratas , Ratas Sprague-Dawley , Sustancia Negra/metabolismo , Sustancia Negra/patología , Sustancia Negra/virología , alfa-Sinucleína/genéticaRESUMEN
In this chapter, we describe a novel ascorbate peroxidase (APEX)-based labeling method that in combination with mass spectrometry identifies proteins in the immediate vicinity of αSyn in living rat cortical neurons. To isolate these interactions, we transduced primary cortical neurons with a lentivirus encoding APEX2 tagged to the C-terminus of alpha-synuclein (αSyn) and under the control of a synapsin promoter. Neural protein lysates were then incubated with streptavidin magnetic beads, washed, eluted from the beads, and digested overnight. The desalted peptides were then labeled with iTRAQ (4-plex) reagents and analyzed by nanoflow liquid chromatography-tandem mass spectrometry (LC-MS/MS). Collected data were analyzed using Spectrum Mill software, ultimately shedding light on αSyn physiological function and abnormal behavior during pathology.
Asunto(s)
Hibridación in Situ , Espectrometría de Masas , Neuronas/metabolismo , Peroxidasa/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Animales , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Análisis de Datos , Técnica del Anticuerpo Fluorescente , Humanos , Enfermedad de Parkinson/metabolismo , Proteómica/métodos , Ratas , Coloración y EtiquetadoRESUMEN
Relative neuronal vulnerability is a universal yet poorly understood feature of neurodegenerative diseases. In Parkinson's disease, dopaminergic (DA) neurons in the substantia nigra (SN) (A9) are particularly vulnerable, whereas adjacent DA neurons within the ventral tegmental area (A10) are essentially spared. Our previous laser capture microdissection and microarray study (Chung et al., 2005) demonstrated that molecular differences between these DA neurons may underlie their differential vulnerability. Here we show that G-substrate, an endogenous inhibitor of Ser/Thr protein phosphatases, exhibits higher expression in A10 compared with A9 DA neurons in both rodent and human midbrain. Overexpression of G-substrate protected dopaminergic BE(2)-M17 cells against toxins, including 6-OHDA and MG-132 (carbobenzoxy-L-leucyl- L-leucyl-L-leucinal), whereas RNA interference (RNAi)-mediated knockdown of endogenous G-substrate increased their vulnerability to these toxins. G-substrate reduced 6-OHDA-mediated protein phosphatase 2A (PP2A) activation in vitro and increased phosphorylated levels of PP2A targets including Akt, glycogen synthase kinase 3beta, and extracellular signal-regulated kinase 2 but not p38. RNAi to Akt diminished the protective effect of G-substrate against 6-OHDA. In vivo, lentiviral delivery of G-substrate to the rat SN increased baseline levels of phosphorylated Akt and protected A9 DA neurons from 6-OHDA-induced toxicity. These results suggest that inherent differences in the levels of G-substrate contribute to the differential vulnerability of DA neurons and that enhancing G-substrate levels may be a neuroprotective strategy for the vulnerable A9 (SN) DA neurons in Parkinson's disease.
Asunto(s)
Proteínas del Tejido Nervioso/fisiología , Enfermedad de Parkinson/enzimología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Leupeptinas/toxicidad , Ratones , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Oxidopamina/toxicidad , Enfermedad de Parkinson/genética , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Proteína Fosfatasa 2 , Interferencia de ARN/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
The lack of disease-modifying treatments for neurodegenerative disease stems in part from our rudimentary understanding of disease mechanisms and the paucity of targets for therapeutic intervention. Here we used an integrated discovery paradigm to identify a new therapeutic target for diseases caused by α-synuclein (α-syn), a small lipid-binding protein that misfolds and aggregates in Parkinson's disease and other disorders. Using unbiased phenotypic screening, we identified a series of compounds that were cytoprotective against α-syn-mediated toxicity by inhibiting the highly conserved enzyme stearoyl-CoA desaturase (SCD). Critically, reducing the levels of unsaturated membrane lipids by inhibiting SCD reduced α-syn toxicity in human induced pluripotent stem cell (iPSC) neuronal models. Taken together, these findings suggest that inhibition of fatty acid desaturation has potential as a therapeutic approach for the treatment of Parkinson's disease and other synucleinopathies.
Asunto(s)
Estearoil-CoA Desaturasa/antagonistas & inhibidores , alfa-Sinucleína/toxicidad , Animales , Citoprotección/efectos de los fármacos , Ácidos Grasos/metabolismo , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oxadiazoles/química , Oxadiazoles/farmacología , Agregado de Proteínas , Ratas , Saccharomyces cerevisiae/efectos de los fármacos , Estearoil-CoA Desaturasa/metabolismo , Triglicéridos/metabolismoRESUMEN
Synucleinopathies, including Parkinson's disease (PD), are associated with the misfolding and mistrafficking of alpha-synuclein (α-syn). Here, using an ascorbate peroxidase (APEX)-based labeling method combined with mass spectrometry, we defined a network of proteins in the immediate vicinity of α-syn in living neurons to shed light on α-syn function. This approach identified 225 proteins, including synaptic proteins, proteins involved in endocytic vesicle trafficking, the retromer complex, phosphatases and mRNA binding proteins. Many were in complexes with α-syn, and some were encoded by genes known to be risk factors for PD and other neurodegenerative diseases. Endocytic trafficking and mRNA translation proteins within this spatial α-syn map overlapped with genetic modifiers of α-syn toxicity, developed in an accompanying study (Khurana et al., this issue of Cell Systems). Our data suggest that perturbation of these particular pathways is directly related to the spatial localization of α-syn within the cell. These approaches provide new avenues to systematically examine protein function and pathology in living cells.