RESUMO
The D620N mutation in vacuolar protein sorting protein 35 (VPS35) gene has been identified to be linked to late onset familial Parkinson disease (PD). However, the pathophysiological roles of VPS35-D620N in PD remain unclear. Here, we generated the transgenic Caenorhabditis elegans overexpressing either human wild type or PD-linked mutant VPS35-D620N in neurons. C. elegans expressing VPS35-D620N, compared with non-transgenic controls, showed movement disorders and dopaminergic neuron loss. VPS35-D620N worms displayed more swimming induced paralysis but showed no defects in BSR assays, thus indicating the disruption of dopamine (DA) recycling back inside neurons. Moreover, VPS35 formed a protein interaction complex with DA transporter (DAT), RAB5, RAB11 and FAM21. In contrast, the VPS35-D620N mutant destabilized these interactions, thus disrupting DAT transport from early endosomes to recycling endosomes, and decreasing DAT at the cell surface. These effects together increased DA in synaptic clefts, and led to dopaminergic neuron degeneration and motor dysfunction. Treatment with reserpine significantly decreased the swimming induced paralysis in VPS35-D620N worms, as compared with vehicle treated VPS35-D620N worms. Our studies not only provide novel insights into the mechanisms of VPS35-D620N-induced dopaminergic neuron degeneration and motor dysfunction via disruption of DAT function and the DA signaling pathway but also indicate a potential strategy to treat VPS35-D620N-related PD and other disorders.
Assuntos
Dopamina , Doença de Parkinson , Animais , Humanos , Dopamina/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Transporte Proteico , Neurônios Dopaminérgicos/metabolismo , Doença de Parkinson/metabolismo , Degeneração Neural/patologia , Paralisia/genética , Paralisia/metabolismo , Paralisia/patologiaRESUMO
Parkinson's disease (PD) is a neurodegenerative disease with movement disorders including resting tremor, rigidity, bradykinesia and postural instability. Recent studies have identified a new PD associated gene, TMEM230 (transmembrane protein 230). However, the pathological roles of TMEM230 and its variants are not fully understood. TMEM230 gene encodes two protein isoforms. Isoform2 is the major protein form (~95%) in human. In this study, we overexpress isoform2 TMEM230 variants (WT or PD-linked *184Wext*5 mutant) or knockdown endogenous protein in cultured SH-5Y5Y cells and mouse primary hippocampus neurons to study their pathological roles. We found that overexpression of WT and mutant TMEM230 or knockdown of endogenous TMEM230-induced neurodegeneration and impaired mitochondria transport at the retrograde direction in axons. Mutant TMEM230 caused more severe neurotoxicity and mitochondrial transport impairment than WT-TMEM230 did. Our results demonstrate that maintaining TMEM230 protein levels is critical for neuron survival and axon transport. These findings suggest that mutant-TMEM230-induced mitochondrial transport impairment could be the early event leading to neurite injury and neurodegeneration in PD development.
Assuntos
Doenças Neurodegenerativas , Doença de Parkinson , Animais , Transporte Axonal/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doença de Parkinson/genéticaRESUMO
Expression of synphilin-1 in neurons induces hyperphagia and obesity in a Drosophila model. However, the molecular pathways underlying synphilin-1-linked obesity remain unclear. Here, Drosophila models and genetic tools were used to study the synphilin-1-linked pathways in energy balance by combining molecular biology and pharmacological approaches. We found that expression of human synphilin-1 in flies increased AMP-activated kinase (AMPK) phosphorylation at Thr172 compared with that in non-transgenic flies. Knockdown of AMPK reduced AMPK phosphorylation and food intake in non-transgenic flies, and further suppressed synphilin-1-induced AMPK phosphorylation, hyperphagia, fat storage and body weight gain in transgenic flies. Expression of constitutively activated AMPK significantly increased food intake and body weight gain in non-transgenic flies, but it did not alter food intake in the synphilin-1 transgenic flies. In contrast, expression of dominant-negative AMPK reduced food intake in both non-transgenic and synphilin-1 transgenic flies. Treatment with STO-609 also suppressed synphilin-1-induced AMPK phosphorylation, hyperphagia and body weight gain. These results demonstrate that the AMPK signaling pathway plays a critical role in synphilin-1-induced hyperphagia and obesity. These findings provide new insights into the mechanisms of synphilin-1-controlled energy homeostasis.
Assuntos
Proteínas Quinases Ativadas por AMP , Proteínas de Transporte/genética , Drosophila , Hiperfagia , Proteínas do Tecido Nervoso/genética , Obesidade , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Animais Geneticamente Modificados , Drosophila/genética , Drosophila/metabolismo , Humanos , Hiperfagia/genética , Obesidade/genética , Transdução de Sinais/genéticaRESUMO
Parkinson's disease (PD) is a common neurodegenerative disease with a heterogeneous etiology that involves genetic and environmental factors or exogenous. Current LRRK2 PD animal models only partly reproduce the characteristics of the disease with very subtle dopaminergic neuron degeneration. We developed a new model of PD that combines a sub-toxic MPTP insult to the G2019S-LRRK2 mutation. Our newly generated mice, overexpressing mutant G2019S-LRRK2 protein in the brain, displayed a mild, age-dependent progressive motor impairment, but no reduction of lifespan. Cortical neurons from G2019S-LRRK2 mice showed an increased vulnerability to stress insults, compared with neurons overexpressing wild-type WT-LRRK2, or non-transgenic (nTg) neurons. The exposure of LRRK2 transgenic mice to a sub-toxic dose of MPTP resulted in severe motor impairment, selective loss of dopamine neurons and increased astrocyte activation, whereas nTg mice with MPTP exposure showed no deficits. Interestingly, mice overexpressing WT-LRRK2 showed a significant impairment that was milder than for the mutant G2019S-LRRK2 mice. L-DOPA treatments could partially improve the movement impairments but did not protect the dopamine neuron loss. In contrast, treatments with an LRRK2 kinase inhibitor significantly reduced the dopaminergic neuron degeneration in this interaction model. Our studies provide a novel LRRK2 gene-MPTP interaction PD mouse model, and a useful tool for future studies of PD pathogenesis and therapeutic intervention.
Assuntos
Dopamina/metabolismo , Neurônios Dopaminérgicos/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Transtornos Motores/patologia , Mutação , Transtornos Parkinsonianos/patologia , Animais , Neurônios Dopaminérgicos/metabolismo , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Transtornos Motores/etiologia , Transtornos Motores/metabolismo , Transtornos Parkinsonianos/etiologia , Transtornos Parkinsonianos/metabolismoRESUMO
Although document studies (including ours) have been reported the achieved in vitro osteoclastic cellular model establishment from the RAW264.7 cell lineage, there was no study directly reported that American Type Culture Collection (ATCC) cell bank has various RAW264.7 cell lineages. Besides that, for our knowledge there was only one study compared the two different RAW264.7TIB-71 and RAW264.7CRL-2278 cell lineages for their osteoclastic differentiation, and they concluded that the RAW264.7CRL-2278 demonstrated to generate much osteoclast than RAW264.7TIB-71 . However, on the contrary to their results we noticed the fusion of RAW264.7TIB-71 in our previous studies was much compromising. Therefore, we try to explore the two cell lineages for their properties in osteoclastic differentiation with an in-depth cellular cytoskeletal study. Our current study has showed that comparing to the RAW264.7CRL-2278 , RAW264.7TIB-71 demonstrated a much higher efficacies for RANKL-stimulated osteoclastic differentiation. Besides that, in our depth cytoskeletal studies, we found that the RANKL-induced RAW264.7TIB-71 cells could finally differentiate into mature osteoclasts. However, regardless the various pre-treatment conditions, there was no mature osteoclast formed in RANKL-induced RAW264.7CRL-2278 cell lineage.
Assuntos
Diferenciação Celular , Proliferação de Células , Citoesqueleto/fisiologia , Osteoclastos/citologia , Ligante RANK/metabolismo , Animais , Camundongos , Osteoclastos/metabolismo , Ligante RANK/genética , Células RAW 264.7RESUMO
Parkinson's disease (PD) is one of the most common movement disorders with loss of dopaminergic neurons and the presence of Lewy bodies in certain brain areas. However, it is not clear how Lewy body (inclusion with protein aggregation) formation occurs. Mutations in leucine-rich repeat kinase 2 (LRRK2) can cause a genetic form of PD and contribute to sporadic PD with the typical Lewy body pathology. Here, we used our recently identified LRRK2 GTP-binding inhibitors as pharmacological probes to study the LRRK2-linked ubiquitination and protein aggregation. Pharmacological inhibition of GTP-binding by GTP-binding inhibitors (68 and Fx2149) increased LRRK2-linked ubiquitination predominantly via K27 linkage. Compound 68- or Fx2149 increased G2019S-LRRK2-linked ubiquitinated aggregates, which occurred through the atypical linkage types K27 and K63. Coexpression of K27R and K63R, which prevented ubiquitination via K27 and K63 linkages, reversed the effects of 68 and Fx2149. Moreover, 68 and Fx2149 also promoted G2019S-LRRK2-linked aggresome (Lewy body-like inclusion) formation via K27 and K63 linkages. These findings demonstrate that LRRK2 GTP-binding activity is critical in LRRK2-linked ubiquitination and aggregation formation. These studies provide novel insight into the LRRK2-linked Lewy body-like inclusion formation underlying PD pathogenesis.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Corpos de Lewy/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Corpos de Lewy/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , UbiquitinaçãoRESUMO
BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disease and the most common movement disorder characterized by motor impairments resulting from midbrain dopamine neuron loss. Abnormalities in small pial arteries and arterioles, which are the primary pathways of local delivery of nutrients and oxygen in brain tissue, have been reported in many neurodegenerative diseases including PD. Mutations in LRRK2 cause genetic PD and contribute to sporadic PD. The most common PD-linked mutation LRRK2 G2019S contributes 20-47% of genetic forms of PD in Caucasian populations. The human LRRK2 G2019S transgenic mouse model displays PD-like movement impairment and was used to identify novel LRRK2 inhibitors, which provides a useful model for studying microvascular abnormalities in PD. OBJECTIVES: To investigate abnormalities in arteriolar cerebral blood volume (CBVa) in various brain regions using the inflow-based vascular-space occupancy (iVASO) MRI technique in LRRK2 mouse models of PD. METHODS: Anatomical and iVASO MRI scans were performed in 5 female and 7 male nontransgenic (nTg), 3 female and 4 male wild-type (WT) LRRK2, and 5 female and 7 male G2019S-LRRK2 mice of 9 months of age. CBVa was calculated and compared in the substantia nigra (SN), olfactory cortex, and prefrontal cortex. RESULTS: Compared to nTg mice, G2019S-LRRK2 mice showed decreased CBVa in the SN, but increased CBVa in the olfactory and prefrontal cortex in both male and female groups, whereas WT-LRRK2 mice showed no change in CBVa in the SN (male and female), the olfactory (female), and prefrontal (female) cortex, but a slight increase in CBVa in the olfactory and prefrontal cortex in the male group only. CONCLUSIONS: Alterations in the blood volume of small arteries and arterioles (CBVa) were detected in the G2019S-LRRK2 mouse model of PD. The opposite changes in CBVa in the SN and the cortex indicate that PD pathology may have differential effects in different brain regions. Our results suggest the potential value of CBVa as a marker for clinical PD studies.
Assuntos
Arteríolas/diagnóstico por imagem , Volume Sanguíneo Cerebral , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Imageamento por Ressonância Magnética , Animais , Arteríolas/patologia , Encéfalo/patologia , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Doença de Parkinson/patologiaRESUMO
A role for the cytoplasmic protein synphilin-1 in regulating energy balance has been demonstrated recently. Expression of synphilin-1 increases ATP levels in cultured cells. However, the mechanism by which synphilin-1 alters cellular energy status is unknown. Here, we used cell models and biochemical approaches to investigate the cellular functions of synphilin-1 on the AMP-activated protein kinase (AMPK) signaling pathway, which may affect energy balance. Overexpression of synphilin-1 increased AMPK phosphorylation (activation). Moreover, synphilin-1 interacted with AMPK by co-immunoprecipitation and GST (glutathione S-transferase) pull-down assays. Knockdown of synphilin-1 reduced AMPK phosphorylation. Overexpression of synphilin-1 also altered AMPK downstream signaling, i.e., a decrease in acetyl CoA carboxylase (ACC) phosphorylation, and an increase in p70S6K phosphorylation. Treatment of compound C (an AMPK inhibitor) reduced synphilin-1 binding with AMPK. In addition, compound C diminished synphilin-1-induced AMPK phosphorylation, and the increase in cellular ATP (adenosine triphosphate) levels. Our results demonstrated that synphilin-1 couples with AMPK, and they exert mutual effects on each other to regulate cellular energy status. These findings not only identify novel cellular actions of synphilin-1, but also provide new insights into the roles of synphilin-1 in regulating energy currency, ATP.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Acetil-CoA Carboxilase/metabolismo , Células HEK293 , Humanos , Fosforilação , Ligação Proteica , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Bone homeostasis is preserved by the balance of maintaining between the activity of osteogenesis and osteoclastogenesis. However, investigations for the osteoclastogenesis were hampered by considerable difficulties associated with isolating and culturing osteoclast in vivo. As the alternative, stimuli-induced osteoclasts formation from RAW264.7 cells (RAW-OCs) have gain its importance for extensively osteoclastogenic study of bone diseases, such as rheumatoid arthritis, osteoporosis, osteolysis and periodontitis. However, considering the RAW-OCs have not yet been well-characterized and RAW264.7 cells are polymorphic because of a diverse phenotype of the individual cells comprising this cell linage, and different fate associated with various stimuli contributions. Thus, in present study, we provide an overview for current knowledge of the phenotype of RAW264.7 cells, as well as the current understanding of the complicated interactions between various stimuli and RAW-OCs in the light of the recent progress.
Assuntos
Artrite Reumatoide/genética , Reabsorção Óssea/genética , Osteogênese/genética , Osteoporose/genética , Animais , Artrite Reumatoide/patologia , Reabsorção Óssea/patologia , Diferenciação Celular/genética , Linhagem da Célula/genética , Humanos , Camundongos , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteoporose/patologia , Células RAW 264.7RESUMO
Melatonin's therapeutic potential has been highly underestimated because its biological functional roles are diverse and relevant mechanisms are complicated. Among the numerous biological activities of melatonin, its regulatory effects on pluripotent mesenchymal stem cells (MSCs), which are found in bone marrow stem cells (BMSCs) and adipose tissue (AD-MSC), have been recently proposed, which has received increasingly more attention in recent studies. Moreover, receptor-dependent and receptor-independent responses to melatonin are identified to occur in these cells by regulating signaling pathways, which drive the commitment and differentiation of MSCs into osteogenic, chondrogenic, or adipogenic lineages. Therefore, the aim of our current review is to summarize the evidence related to the utility of melatonin as a regulatory agent by focusing on its relationship with the differentiation of MSCs. In particular, we aimed to review its roles in promoting osteogenic and chondrogenic differentiation and the relevant signaling cascades involved. Also, the roles that melatonin and, particularly, its receptors play in these processes are highlighted.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Melatonina/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Animais , Células da Medula Óssea/metabolismo , Linhagem da Célula , Condrogênese/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteogênese/efeitos dos fármacos , Transdução de SinaisRESUMO
Established RAW264.7 cell lines for osteoclastic differentiation has been widely engaged in bone homeostasis research, however, the efficacy of RANKL independently stimulating has rarely been defined, because protocols were usually developed and modified by various laboratories. Otherwise, problematic issues are also lie in the cell's seeding density, RANKL stimulating time point, and distinguishing osteoclastogenesis ability of RANKL-treated RAW264.7 cells. Therefore, in the current study, we examined the efficacy of various concentrations of RANKL-treated RAW264.7 for its osteoclastic differentiation with or without pretreated other costimulators such as: LPS and/or M-CSF. The oteoclastogenesis ability of RANKL-treated RAW264.7 cells was demonstrated by bone resorption pit, F-actin, and osteoclastogenesis specific marker studies. Besides that, through tartrate-resistant acid phosphatase (TRAP) staining, we clarified to start the treatment with 30 ng/ml RANKL at 12 hr after seeded RAW264.7 with the density of 6.25 × 10 3 cells/cm 2 manifested an significantly increased number of multinucleated osteoclastic cells. Overall, our results establishing an optimal method for RANKL independently inducing RAW 264.7 cell osteoclastic differentiation, which could efficiently generate osteoclasts in vitro for significant advances in our understanding of bone biology.
Assuntos
Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Osteoclastos/metabolismo , Ligante RANK/farmacologia , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Animais , Diferenciação Celular/fisiologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Osteoclastos/efeitos dos fármacos , Osteogênese , Células RAW 264.7RESUMO
Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal-dominant Parkinsonism with pleomorphic pathology including deposits of aggregated protein and neuronal degeneration. The pathogenesis of LRRK2-linked Parkinson's disease (PD) is not fully understood. Here, using co-immunoprecipitation, we found that LRRK2 interacted with synphilin-1 (SP1), a cytoplasmic protein that interacts with α-synuclein and has implications in PD pathogenesis. LRRK2 interacted with the N-terminus of SP1 whereas SP1 predominantly interacted with the C-terminus of LRRK2, including kinase domain. Co-expression of SP1 with LRRK2 increased LRRK2-induced cytoplasmic aggregation in cultured cells. Moreover, SP1 also attenuated mutant LRRK2-induced toxicity and reduced LRRK2 kinase activity in cultured cells. Knockdown of SP1 by siRNA enhanced LRRK2 neuronal toxicity. In vivo Drosophila studies, co-expression of SP1 and mutant G2019S-LRRK2 in double transgenic Drosophila increased survival and improved locomotor activity. Expression of SP1 protects against G2019S-LRRK2-induced dopamine neuron loss and reduced LRRK2 phosphorylation in double transgenic fly brains. Our findings demonstrate that SP1 attenuates mutant LRRK2-induced PD-like phenotypes and plays a neural protective role.
Assuntos
Proteínas de Transporte/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Doença de Parkinson/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Dopamina/metabolismo , Drosophila , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Camundongos , Mutação , Degeneração Neural/genética , Proteínas do Tecido Nervoso/genética , Neurônios/enzimologia , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Fosforilação , Domínios e Motivos de Interação entre Proteínas , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Although the pathogenesis of Parkinson's disease (PD) remains unclear, mutations in leucine-rich repeat kinase 2 (Lrrk2) are among the major causes of familial PD. Most of these mutations disrupt Lrrk2 kinase and (or) GTPase domain function, resulting in neuronal degeneration. However, the signal pathways underlying Lrrk2-induced neuronal degeneration are not fully understood. There is an expanding body of evidence that suggests a link between Lrrk2 function and MAP kinase (MAPK) cascades. To further investigate this link in vivo, genetic RNAi screens of the MAPK pathways were performed in a Drosophila model to identify genetic modifier(s) that can suppress G2019S-Lrrk2-induced PD-like phenotypes. The results revealed that the knockdown of hemipterous (hep, or JNKK) increased fly survival time, improved locomotor function, and reduced loss of dopaminergic neurons in G2019S-Lrrk2 transgenic flies. Expression of the dominant-negative allele of JNK (JNK-DN), a kinase that is downstream of hep in G2019S-Lrrk2 transgenic flies, elicited a similar effect. Moreover, treatment with the JNK inhibitor SP600125 partially reversed the G2019S-Lrrk2-induced loss of dopaminergic neurons. These results indicate that the hep pathway plays an important role in Lrrk2-linked Parkinsonism in flies. These studies provide new insights into the molecular mechanisms underlying Lrrk2-linked PD pathogenesis and aid in identifying potential therapeutic targets.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Proteínas de Drosophila/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Doenças Neurodegenerativas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Drosophila melanogaster , Mutação/genética , Transdução de Sinais/fisiologiaRESUMO
Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause autosomal-dominant Parkinson's disease (PD) and contribute to sporadic PD. LRRK2 contains Guanosine-5'-triphosphate (GTP) binding, GTPase and kinase activities that have been implicated in the neuronal degeneration of PD pathogenesis, making LRRK2, a potential drug target. To date, there is no disease-modifying drug to slow the neuronal degeneration of PD and no published LRRK2 GTP domain inhibitor. Here, the biological functions of two novel GTP-binding inhibitors of LRRK2 were examined in PD cell and mouse models. Through a combination of computer-aided drug design (CADD) and LRRK2 bio-functional screens, two novel compounds, 68: and 70: , were shown to reduce LRRK2 GTP binding and to inhibit LRRK2 kinase activity in vitro and in cultured cell assays. Moreover, these two compounds attenuated neuronal degeneration in human SH-SY5Y neuroblastoma cells and mouse primary neurons expressing mutant LRRK2 variants. Although both compounds inhibited LRRK2 kinase activity and reduced neuronal degeneration, solubility problems with 70: prevented further testing in mice. Thus, only 68: was tested in a LRRK2-based lipopolysaccharide (LPS)-induced pre-inflammatory mouse model. 68: reduced LRRK2 GTP-binding activity and kinase activity in brains of LRRK2 transgenic mice after intraperitoneal injection. Moreover, LPS induced LRRK2 upregulation and microglia activation in mouse brains. These findings suggest that disruption of GTP binding to LRRK2 represents a potential novel therapeutic approach for PD intervention and that these novel GTP-binding inhibitors provide both tools and lead compounds for future drug development.
Assuntos
Guanosina Trifosfato/metabolismo , Neurônios/efeitos dos fármacos , Doença de Parkinson/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Sulfonas/farmacologia , Tiazóis/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Humanos , Inflamação/metabolismo , Inflamação/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Mutação , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/enzimologia , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Sulfonas/uso terapêutico , Tiazóis/uso terapêuticoRESUMO
Aquaporin-4 (AQP4) is essential for normal functioning of the brain's glymphatic system. Impaired glymphatic function is associated with neuroinflammation. Recent clinical evidence suggests the involvement of glymphatic dysfunction in LRRK2-associated Parkinson's disease (PD); however, the precise mechanism remains unclear. The pro-inflammatory cytokine interferon (IFN) γ interacts with LRRK2 to induce neuroinflammation. Therefore, we examined the AQP4-dependent glymphatic system's role in IFNγ-mediated neuroinflammation in LRRK2-associated PD. We found that LRRK2 interacts with and phosphorylates AQP4 in vitro and in vivo. AQP4 phosphorylation by LRRK2 R1441G induced AQP4 depolarization and disrupted glymphatic IFNγ clearance. Exogeneous IFNγ significantly increased astrocyte expression of IFNγ receptor, amplified AQP4 depolarization, and exacerbated neuroinflammation in R1441G transgenic mice. Conversely, inhibiting LRRK2 restored AQP4 polarity, improved glymphatic function, and reduced IFNγ-mediated neuroinflammation and dopaminergic neurodegeneration. Our findings establish a link between LRRK2-mediated AQP4 phosphorylation and IFNγ-mediated neuroinflammation in LRRK2-associated PD, guiding the development of LRRK2 targeting therapy.
RESUMO
Alzheimer's disease (AD) is the most common neurodegenerative disease, with sporadic form being the predominant type. Neuroinflammation plays a critical role in accelerating pathogenic processes in AD. Mesenchymal stem cell (MSC)-derived small extracellular vesicles (MSC-sEVs) regulate inflammatory responses and show great promise for treating AD. Induced pluripotent stem cell (iPSC)-derived MSCs are similar to MSCs and exhibit low immunogenicity and heterogeneity, making them promising cell sources for clinical applications. This study examined the anti-inflammatory effects of MSC-sEVs in a streptozotocin-induced sporadic mouse model of AD (sAD). The intracisternal administration of iPSC-MSC-sEVs alleviated NLRP3/GSDMD-mediated neuroinflammation, decreased amyloid deposition and neuronal apoptosis, and mitigated cognitive dysfunction. Furthermore, it explored the role of miR-223-3p in the iPSC-MSC-sEVs-mediated anti-inflammatory effects in vitro. miR-223-3p directly targeted NLRP3, whereas inhibiting miR-223-3p almost completely reversed the suppression of NLRP3 by MSC-sEVs, suggesting that miR-223-3p may, at least partially, account for MSC-sEVs-mediated anti-inflammation. Results obtained suggest that intracisternal administration of iPSC-MSC-sEVs can reduce cognitive impairment by inhibiting NLRP3/GSDMD neuroinflammation in a sAD mouse model. Therefore, the present study provides a proof-of-principle for applying iPSC-MSC-sEVs to target neuroinflammation in sAD.
Assuntos
Doença de Alzheimer , Modelos Animais de Doenças , Vesículas Extracelulares , Células-Tronco Mesenquimais , MicroRNAs , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Camundongos , Doença de Alzheimer/patologia , Doença de Alzheimer/terapia , Doença de Alzheimer/metabolismo , Vesículas Extracelulares/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Inflamação/patologia , Inflamação/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , MicroRNAs/genética , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismoRESUMO
Mutations in leucine-rich repeat kinase 2 (LRRK2) have been identified as a genetic cause of familial Parkinson's disease (PD) and have also been found in the more common sporadic form of PD, thus positioning LRRK2 as important in the pathogenesis of PD. Biochemical studies of the disease-causing mutants of LRRK2 implicates an enhancement of kinase activity as the basis of neuronal toxicity and thus possibly the pathogenesis of PD due to LRRK2 mutations. Previously, a chemical library screen identified inhibitors of LRRK2 kinase activity. Here, two of these inhibitors, GW5074 and sorafenib, are shown to protect against G2019S LRRK2-induced neurodegeneration in vivo in Caenorhabditis elegans and in Drosophila. These findings indicate that increased kinase activity of LRRK2 is neurotoxic and that inhibition of LRRK2 activity can have a disease-modifying effect. This suggests that inhibition of LRRK2 holds promise as a treatment for PD.
Assuntos
Doença de Parkinson/enzimologia , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Animais Geneticamente Modificados , Benzenossulfonatos/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Drosophila/efeitos dos fármacos , Drosophila/genética , Drosophila/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Humanos , Indóis/farmacologia , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Mutação/genética , Niacinamida/análogos & derivados , Oxidopamina/efeitos adversos , Doença de Parkinson/genética , Doença de Parkinson/prevenção & controle , Fenóis/farmacologia , Compostos de Fenilureia , Proteínas Serina-Treonina Quinases/genética , Piridinas/farmacologia , Sorafenibe , Sinucleínas/efeitos adversosRESUMO
Parkinson's disease (PD) is a neurodegenerative disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. The pathogenesis of PD is believed to involve both genetic susceptibility and environmental factors. Mutations in Leucine-rich repeat kinase 2 (LRRK2) cause genetic forms of PD, and the LRRK2 locus contributes to sporadic PD. Environmental toxins are believed to act in part by causing oxidative stress. Here we employed cell and Drosophila models to investigate the interaction between LRRK2 genetic mutations and oxidative stress. We found that H(2)O(2) increased LRRK2 kinase activity and enhanced LRRK2 cell toxicity in cultured cells and mouse primary cortical neurons. Furthermore, a sub-toxic dose of H(2)O(2) significantly shortened the survival of LRRK2 transgenic flies and augmented LRRK2-induced locomotor defects and dopamine neuron loss. Treatment with a LRRK2 kinase inhibitor (GW5074) or an anti-oxidant (curcumin) significantly suppressed these PD-like phenotypes in flies. Moreover, curcumin significantly reduced LRRK2 kinase activity and the levels of oxidized proteins, and thus acted as not only an antioxidant but also a LRRK2 kinase inhibitor. These results indicate that LRRK2 genetic alterations can interact with oxidative stress, converging on a pathogenic pathway that may be related to PD. These studies also identified curcumin as a LRRK2 kinase inhibitor that may be a useful candidate for LRRK2-linked PD intervention.
Assuntos
Curcumina/farmacologia , Proteínas de Drosophila/genética , Inibidores Enzimáticos/farmacologia , Mutação/genética , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Análise de Variância , Animais , Animais Geneticamente Modificados , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Córtex Cerebral/citologia , Dopamina/metabolismo , Drosophila , Proteínas de Drosophila/metabolismo , Interações Medicamentosas , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Imunoprecipitação , Indóis/farmacologia , Camundongos , Modelos Animais , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Fenóis/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Análise de Sobrevida , TransfecçãoRESUMO
Genetic alterations in alpha-synuclein cause autosomal dominant familial Parkinsonism and may contribute to sporadic Parkinson's disease (PD). Synphilin-1 is an alpha-synuclein-interacting protein, with implications in PD pathogenesis related to protein aggregation. Currently, the in vivo role of synphilin-1 in alpha-synuclein-linked pathogenesis is not fully understood. Using the mouse prion protein promoter, we generated synphilin-1 transgenic mice, which did not display PD-like phenotypes. However, synphilin-1/A53T alpha-synuclein double-transgenic mice survived longer than A53T alpha-synuclein single-transgenic mice. There were attenuated A53T alpha-synuclein-induced motor abnormalities and decreased astroglial reaction and neuronal degeneration in brains in double-transgenic mice. Overexpression of synphilin-1 decreased caspase-3 activation, increased beclin-1 and LC3 II expression and promoted formation of aggresome-like structures, suggesting that synphilin-1 alters multiple cellular pathways to protect against neuronal degeneration. These studies demonstrate that synphilin-1 can diminish the severity of alpha-synucleinopathy and play a neuroprotective role against A53T alpha-synuclein toxicity in vivo.
Assuntos
Encéfalo/patologia , Proteínas de Transporte/genética , Degeneração Neural/metabolismo , Proteínas do Tecido Nervoso/genética , Doença de Parkinson/genética , alfa-Sinucleína/genética , Análise de Variância , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Beclina-1 , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Caspase 3/metabolismo , Immunoblotting , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Corpos de Lewy/metabolismo , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto/genética , Degeneração Neural/etiologia , Proteínas do Tecido Nervoso/metabolismo , Doença de Parkinson/complicaçõesRESUMO
Mutation in leucine-rich repeat kinase-2 (LRRK2) is the most common cause of late-onset Parkinson's disease (PD). Although most cases of PD are sporadic, some are inherited, including those caused by LRRK2 mutations. Because these mutations may be associated with a toxic gain of function, controlling the expression of LRRK2 may decrease its cytotoxicity. Here we show that the carboxyl terminus of HSP70-interacting protein (CHIP) binds, ubiquitinates, and promotes the ubiquitin proteasomal degradation of LRRK2. Overexpression of CHIP protects against and knockdown of CHIP exacerbates toxicity mediated by mutant LRRK2. Moreover, HSP90 forms a complex with LRRK2, and inhibition of HSP90 chaperone activity by 17AAG leads to proteasomal degradation of LRRK2, resulting in increased cell viability. Thus, increasing CHIP E3 ligase activity and blocking HSP90 chaperone activity can prevent the deleterious effects of LRRK2. These findings point to potential treatment options for LRRK2-associated PD.