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2.
Brain ; 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38976650

RESUMO

Mutations in the GBA1 gene are common genetic risk factors for Parkinson's disease (PD), disrupting enzymatic activity and causing lysosomal dysfunction, leading to elevated α-synuclein (α-syn) levels. While GBA1's role in synucleinopathy is well-established, recent research underscores neuroinflammation as a significant pathogenic mechanism in GBA1 deficiency. This study investigates neuroinflammation in Gba1 E326K knock-in mice, a model associated with increased PD and dementia risk. At 9 and 24 months, we assessed GBA1 protein and activity, α-synuclein pathology, neurodegeneration, motor deficits, and gliosis in the ventral midbrain and hippocampus using immunohistochemistry (IHC), Western blot (WB), and GCase assays. Additionally, primary microglia from WT and GBA1E326K/E326K mice were treated with α-syn preformed fibrils (PFF) to study microglia activation, pro-inflammatory cytokines, reactive astrocyte formation, and neuronal death through qPCR, WB, and immunocytochemistry analyses. We also evaluated the effects of gut inoculation of α-syn PFF in Gba1 E326K mice at 7 months and striatal inoculation at 10 months, assessing motor/non-motor symptoms, α-syn pathology, neuroinflammation, gliosis, and neurodegeneration via behavioural tests, IHC, and WB assays. At 24 months, Gba1 E326K knock-in mice showed reduced GCase enzymatic activity and glucosylceramide build-up in the ventral midbrain and hippocampus. Increased pro-inflammatory cytokines and reactive astrocytes were observed in microglia and astrocytes from Gba1 E326K mice treated with pathologic α-syn PFF. Gut inoculation of α-syn PFF increased Lewy body accumulation in the hippocampal dentate gyrus, with heightened microglia and astrocyte activation and worsened non-motor symptoms. Intrastriatal α-syn preformed fibril injection induced motor deficits, reactive glial protein accumulation, and tauopathy in the prefrontal cortex and hippocampus of Gba1 E326K mice. GBA1 deficiency due to the Gba1 E326K mutation exacerbates neuroinflammation and promotes pathogenic α-synuclein transmission, intensifying disease pathology in PD models. This study enhances our understanding of how the Gba1 E326K mutation contributes to neuroinflammation and the spread of pathogenic α-syn in the brain, suggesting new therapeutic strategies for PD and related synucleinopathies.

3.
Nat Commun ; 15(1): 4663, 2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38821932

RESUMO

Pathologic α-synuclein (α-syn) spreads from cell-to-cell, in part, through binding to the lymphocyte-activation gene 3 (Lag3). Here we report that amyloid ß precursor-like protein 1 (Aplp1) interacts with Lag3 that facilitates the binding, internalization, transmission, and toxicity of pathologic α-syn. Deletion of both Aplp1 and Lag3 eliminates the loss of dopaminergic neurons and the accompanying behavioral deficits induced by α-syn preformed fibrils (PFF). Anti-Lag3 prevents the internalization of α-syn PFF by disrupting the interaction of Aplp1 and Lag3, and blocks the neurodegeneration induced by α-syn PFF in vivo. The identification of Aplp1 and the interplay with Lag3 for α-syn PFF induced pathology deepens our insight about molecular mechanisms of cell-to-cell transmission of pathologic α-syn and provides additional targets for therapeutic strategies aimed at preventing neurodegeneration in Parkinson's disease and related α-synucleinopathies.


Assuntos
Proteína do Gene 3 de Ativação de Linfócitos , alfa-Sinucleína , Animais , Feminino , Humanos , Masculino , Camundongos , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Antígenos CD/metabolismo , Antígenos CD/genética , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doença de Parkinson/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Ligação Proteica
4.
bioRxiv ; 2023 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-37745332

RESUMO

Mutations in the GBA1 gene have been identified as a prevalent genetic risk factor for Parkinson's disease (PD). GBA1 mutations impair enzymatic activity, leading to lysosomal dysfunction and elevated levels of α-synuclein (α-syn). While most research has primarily focused on GBA1's role in promoting synucleinopathy, emerging evidence suggests that neuroinflammation may be a key pathogenic alteration caused by GBA1 deficiency. To examine the molecular mechanism underlying GBA1 deficiency-mediated neuroinflammation, we generated Gba1 E326K knock-in (KI) mice using the CRISPR/Cas9 technology, which is linked to an increased risk of PD and dementia with Lewy bodies (DLB). In the ventral midbrain and hippocampus of 24-month-old Gba1 E326K KI mice, we found a moderate decline in GBA1 enzymatic activity, a buildup of glucosylceramide, and an increase in microglia density. Furthermore, we observed increased levels of pro-inflammatory cytokines and formation of reactive astrocytes in primary microglia and astrocytes, respectively, cultured from Gba1 E326K KI mice following treatment with pathologic α-syn preformed fibrils (PFF). Additionally, the gut inoculation of α-syn PFF in Gba1 E326K KI mice significantly enhanced the accumulation of Lewy bodies in the dentate gyrus of the hippocampus, accompanied by aggravated neuroinflammation and exacerbated non-motor symptoms. This research significantly enhances our understanding of the Gba1 E326K mutation's involvement in neuroinflammation and the cell-to-cell transmission of pathogenic α-syn in the brain, thereby opening new therapeutic avenues.

5.
Cell Stem Cell ; 30(7): 973-986.e11, 2023 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-37339636

RESUMO

Human induced pluripotent stem cells (hiPSCs) offer advantages for disease modeling and drug discovery. However, recreating innate cellular pathologies, particularly in late-onset neurodegenerative diseases with accumulated protein aggregates including Parkinson's disease (PD), has been challenging. To overcome this barrier, we developed an optogenetics-assisted α-synuclein (α-syn) aggregation induction system (OASIS) that rapidly induces α-syn aggregates and toxicity in PD hiPSC-midbrain dopaminergic neurons and midbrain organoids. Our OASIS-based primary compound screening with SH-SY5Y cells identified 5 candidates that were secondarily validated with OASIS PD hiPSC-midbrain dopaminergic neurons and midbrain organoids, leading us to finally select BAG956. Furthermore, BAG956 significantly reverses characteristic PD phenotypes in α-syn preformed fibril models in vitro and in vivo by promoting autophagic clearance of pathological α-syn aggregates. Following the FDA Modernization Act 2.0's emphasis on alternative non-animal testing methods, our OASIS can serve as an animal-free preclinical test model (newly termed "nonclinical test") for the synucleinopathy drug development.


Assuntos
Células-Tronco Pluripotentes Induzidas , Neuroblastoma , Doença de Parkinson , Humanos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Neurônios Dopaminérgicos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Optogenética , Doença de Parkinson/genética
6.
Cells ; 11(24)2022 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-36552734

RESUMO

Non-receptor tyrosine kinase, c-Abl plays a role in the pathogenesis of several neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Here, we found that TDP-43, which was one of the main proteins comprising pathological deposits in amyotrophic lateral sclerosis (ALS), is a novel substrate for c-Abl. The phosphorylation of tyrosine 43 of TDP-43 by c-Abl led to increased TDP-43 levels in the cytoplasm and increased the formation of G3BP1-positive stress granules in SH-SY5Y cells. The kinase-dead mutant of c-Abl had no effect on the cytoplasmic localization of TDP-43. The expression of phosphor-mimetic mutant Y43E of TDP-43 in primary cortical neurons accumulated the neurite granule. Furthermore, the phosphorylation of TDP-43 at tyrosine 43 by c-Abl promoted the aggregation of TDP-43 and increased neuronal cell death in primary cortical neurons, but not in c-Abl-deficient primary cortical neurons. Identification of c-Abl as the kinase of TDP43 provides new insight into the pathogenesis of ALS.


Assuntos
Esclerose Lateral Amiotrófica , Proteínas Proto-Oncogênicas c-abl , Humanos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Neuroblastoma , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Tirosina/metabolismo
8.
Neuron ; 109(23): 3758-3774.e11, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34644545

RESUMO

Impairment in glucocerebrosidase (GCase) is strongly associated with the development of Parkinson's disease (PD), yet the regulators responsible for its impairment remain elusive. In this paper, we identify the E3 ligase Thyroid Hormone Receptor Interacting Protein 12 (TRIP12) as a key regulator of GCase. TRIP12 interacts with and ubiquitinates GCase at lysine 293 to control its degradation via ubiquitin proteasomal degradation. Ubiquitinated GCase by TRIP12 leads to its functional impairment through premature degradation and subsequent accumulation of α-synuclein. TRIP12 overexpression causes mitochondrial dysfunction, which is ameliorated by GCase overexpression. Further, conditional TRIP12 knockout in vitro and knockdown in vivo promotes the expression of GCase, which blocks α-synuclein preformed fibrils (α-syn PFFs)-provoked dopaminergic neurodegeneration. Moreover, TRIP12 accumulates in human PD brain and α-synuclein-based mouse models. The identification of TRIP12 as a regulator of GCase provides a new perspective on the molecular mechanisms underlying dysfunctional GCase-driven neurodegeneration in PD.


Assuntos
Proteínas de Transporte/metabolismo , Glucosilceramidase , Doença de Parkinson , Ubiquitina-Proteína Ligases/metabolismo , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Camundongos , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Ubiquitinação , alfa-Sinucleína/metabolismo
9.
J Med Chem ; 64(20): 15091-15110, 2021 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-34583507

RESUMO

Parkinson's disease (PD) is a progressive neurodegenerative disorder that affects movement. The nonreceptor tyrosine kinase c-Abl has shown a potential role in the progression of PD. As such, c-Abl inhibition is a promising candidate for neuroprotection in PD and α-synucleinopathies. Compound 5 is a newly synthesized blood-brain barrier penetrant c-Abl inhibitor with higher efficacy than existing inhibitors. The objective of the current study was to demonstrate the neuroprotective effects of compound 5 on the α-synuclein preformed fibril (α-syn PFF) mouse model of PD. Compound 5 significantly reduced neurotoxicity, activation of c-Abl, and Lewy body pathology caused by α-syn PFF in cortical neurons. Additionally, compound 5 markedly ameliorated the loss of dopaminergic neurons, c-Abl activation, Lewy body pathology, neuroinflammatory responses, and behavioral deficits induced by α-syn PFF injection in vivo. Taken together, these results suggest that compound 5 could be a pharmaceutical agent to prevent the progression of PD and α-synucleinopathies.


Assuntos
Fármacos Neuroprotetores/farmacologia , Doença de Parkinson/tratamento farmacológico , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Fármacos Neuroprotetores/química , Doença de Parkinson/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Relação Estrutura-Atividade
10.
Genetics ; 209(3): 815-828, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29724861

RESUMO

Post-translational control is a crucial mechanism for circadian timekeeping. Evolutionarily conserved kinases and phosphatases have been implicated in circadian phosphorylation and the degradation of clock-relevant proteins, which sustain high-amplitude rhythms with 24-hr periodicity in animal behaviors and physiology. Here, we report a novel clock function of the heterodimeric Ca2+/calmodulin-dependent phosphatase calcineurin and its regulator sarah (sra) in Drosophila Genomic deletion of the sra locus dampened circadian locomotor activity rhythms in free-running constant dark after entrainment in light-dark cycles. Poor rhythms in sra mutant behaviors were accompanied by lower expression of two oscillating clock proteins, PERIOD (PER) and TIMELESS (TIM), at the post-transcriptional level. RNA interference-mediated sra depletion in circadian pacemaker neurons was sufficient to phenocopy loss-of-function mutation in sra On the other hand, a constitutively active form of the catalytic calcineurin subunit, Pp2B-14DACT, shortened circadian periodicity in locomotor behaviors and phase-advanced PER and TIM rhythms when overexpressed in clock neurons. Heterozygous sra deletion induced behavioral arrhythmicity in Pp2B-14DACT flies, whereas sra overexpression rescued short periods in these animals. Finally, pharmacological inhibition of calcineurin in either wild-type flies or clock-less S2 cells decreased the levels of PER and TIM, likely by facilitating their proteasomal degradation. Taken together, these data suggest that sra negatively regulates calcineurin by cell-autonomously titrating calcineurin-dependent stabilization of PER and TIM proteins, thereby sustaining high-amplitude behavioral rhythms in Drosophila.


Assuntos
Calcineurina/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Circadianas Period/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Linhagem Celular , Ritmo Circadiano , Drosophila/genética , Drosophila/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Circadianas Period/genética , Processamento de Proteína Pós-Traducional , Proteólise
11.
Arch Pharm Res ; 38(11): 2020-8, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25956697

RESUMO

Sesquiterpene lactone compounds have received considerable attention in pharmacological research due to their therapeutic effects including anti-cancer and anti-inflammatory activities. In this report, we investigated the effect of arsantin, a sesquiterpene lactone compound present in Artemisia santolina, on cellular differentiation in the human promyelocytic leukemia HL-60 cell culture system. Arsantin significantly induced HL-60 cell differentiation in a concentration-dependent manner. Cytofluorometric analysis indicated that arsantin induced HL-60 cell differentiation predominantly into granulocytes. Both PKC and MAPK inhibitors suppressed the HL-60 cell differentiation induced by arsantin. Moreover, treatment with arsantin increased protein levels of PKCα and PKCßII isoforms, and also induced increased protein levels and phosphorylation form of MAPKs in HL-60 cells. Importantly, arsantin synergistically enhanced differentiation of HL-60 cells in a dose-dependent manner when combined with either low doses of 1,25-(OH)2D3 or ATRA. The ability to enhance the differentiation potential of 1,25-(OH)2D3 or ATRA by arsantin may improve outcomes in the therapy of acute promyelocytic leukemia.


Assuntos
Artemisia/química , Diferenciação Celular/efeitos dos fármacos , Lactonas/farmacologia , Leucemia Promielocítica Aguda/tratamento farmacológico , Sesquiterpenos/farmacologia , Calcitriol/administração & dosagem , Calcitriol/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Células HL-60 , Humanos , Lactonas/administração & dosagem , Lactonas/isolamento & purificação , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C/metabolismo , Sesquiterpenos/administração & dosagem , Sesquiterpenos/isolamento & purificação , Tretinoína/administração & dosagem , Tretinoína/farmacologia
12.
Arch Pharm Res ; 34(2): 191-8, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21380800

RESUMO

Induction of differentiation is a new and promising approach to leukemia therapy, well illustrated by the treatment of acute promyelocytic leukemia with 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] or all-trans retinoic acid (ATRA). Using combination of either 1,25-(OH)(2)D(3) or ATRA and chemotherapy, adverse effects 1,25-(OH)(2)D(3) or ATRA such as hypercalcemic effects have decreased, and long-term survival has improved. In a previous study, we demonstrated that santonin could be chemically modified into a diacetoxy acetal derivative of santonin with strong differentiation-inducing activity. In this study, we further synthesized C(6)-epimer derivatives of diacetoxy acetal derivative of santonin and tested their effects on HL-60 cell differentiation. Some of the C(6)-epimer derivatives themselves induced increases in cell differentiation. Especially, (11S)-3,3-(ethylenedioxy) eudesmano-13-ol-6ß-acetate (7) was demonstrated to induce differentiation with larger than 80% of the cells attaining a differentiated phenotype. Importantly, 7 strongly enhanced differentiation of HL-60 cells in a dose-dependent manner when combined with either low doses of 1,25-(OH)(2)D(3) or ATRA. The ability to enhance the differentiation potential of 1,25-(OH)(2)D(3) or ATRA by 7 may improve outcomes in the therapy of acute promyelocytic leukemia.


Assuntos
Acetais/síntese química , Acetais/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Leucemia Promielocítica Aguda/tratamento farmacológico , Santonina/análogos & derivados , Acetais/análise , Antineoplásicos/análise , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Células HL-60 , Humanos , Santonina/síntese química , Santonina/química , Santonina/farmacologia , Tretinoína/farmacologia , Vitamina D/análogos & derivados , Vitamina D/farmacologia
13.
Biochem Biophys Res Commun ; 395(1): 104-10, 2010 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-20350534

RESUMO

Chemo-resistance to anti-cancer drugs is a major obstacle in efforts to develop a successful treatment of acute myeloid leukemia (AML). In this study, we investigate whether resveratrol, a common ingredient in a broad variety of fruits and vegetables, can reverse drug resistance in AML cells. Three doxorubicin-resistant AML cell lines (AML-2/DX30, AML-2/DX100, AML-2/DX300) were prepared via long-term exposure to doxorubicin for more than 3 months. DNA microarray analysis demonstrated that many genes were differentially expressed in the resistant cells, as compared with the wild type AML-2/WT cells. In particular, the expression level of the MRP1 gene was significantly increased in the AML-2/DX300 cells, as compared to that detected in AML-2 cells. Importantly, the resveratrol was shown not only to induce cell growth arrest and apoptotic death in doxorubicin-resistant AML cells, but was also shown to downregulate the expression of an MRP1 gene. Furthermore, resveratrol treatment induced a significant increase in the uptake of 5(6)-carboxyfluorescein diacetate, a MRP1 substrate, into the doxorubicin-resistant AML-2/DX300 cells. The results of this study show that resveratrol may facilitate the cellular uptake of doxorubicin via an induced downregulation of MRP1 expression, and also suggest that it may prove useful in overcoming doxorubicin resistance, or in sensitizing doxorubicin-resistant AML cells to anti-leukemic agents.


Assuntos
Doxorrubicina/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Estilbenos/farmacologia , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Fluoresceínas/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Resveratrol
14.
Int J Oncol ; 34(4): 1165-71, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19287976

RESUMO

Resistance to cytarabine (Ara-C) incapacitates the therapeutic effort during the treatment of acute myeloid leukemia (AML). To elucidate mechanism responsible for the development of resistance to Ara-C, we established the Ara-C resistant AML-2/WT cell sublines, AML-2/IDAC and AML-2/ARC. We then conducted DNA microarray analysis to compare the AML-2/IDAC cells with parental AML-2/WT cells. The results of the microarray analysis revealed a severe defect in the expression of deoxycytidine kinase (dCK), which plays a key role in the transformation of Ara-C to the active form in AML-2/IDAC cells. A similar event was observed in AML-2/ARC cells, but not in Ara-C sensitive AML-2/IDA cells that were resistant to idarubicin. The decreased expression of dCK also resulted in lower activity in both Ara-C resistant variants. However, no significant difference in the intracellular concentration of Ara-C was observed among the cells tested, which indicates that the Ara-C resistant phenotype in our models occurred due to the lower expression and activity of dCK rather than a change in the ability to take up Ara-C. Additionally, in vitro assays using BM cells from AML patients revealed that the expression of dCK and the sensitivity to Ara-C were correlated. Taken together, these findings demonstrate that dCK can regulate the in vitro cellular response to Ara-C in AML cells.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Citarabina/farmacologia , Desoxicitidina Quinase/biossíntese , Resistencia a Medicamentos Antineoplásicos , Regulação Enzimológica da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/tratamento farmacológico , Linhagem Celular Tumoral , Fragmentação do DNA , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo
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