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1.
Sci Adv ; 10(6): eadj4767, 2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38335281

RESUMO

Alpha-synuclein (αSyn) protein levels correlate with the risk and severity of Parkinson's disease and related neurodegenerative diseases. Lowering αSyn is being actively investigated as a therapeutic modality. Here, we systematically map the regulatory network that controls endogenous αSyn using sequential CRISPR-knockout and -interference screens in an αSyn gene (SNCA)-tagged cell line and induced pluripotent stem cell-derived neurons (iNeurons). We uncover αSyn modifiers at multiple regulatory layers, with amino-terminal acetyltransferase B (NatB) enzymes being the most potent endogenous αSyn modifiers in both cell lines. Amino-terminal acetylation protects the cytosolic αSyn from rapid degradation by the proteasome in a Ube2w-dependent manner. Moreover, we show that pharmacological inhibition of methionyl-aminopeptidase 2, a regulator of NatB complex formation, attenuates endogenous αSyn in iNeurons carrying SNCA triplication. Together, our study reveals several gene networks that control endogenous αSyn, identifies mechanisms mediating the degradation of nonacetylated αSyn, and illustrates potential therapeutic pathways for decreasing αSyn levels in synucleinopathies.


Assuntos
Acetiltransferase N-Terminal B , Doença de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Neurônios/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Acetiltransferase N-Terminal B/antagonistas & inibidores , Acetiltransferase N-Terminal B/metabolismo , Metionil Aminopeptidases/antagonistas & inibidores , Metionil Aminopeptidases/metabolismo
2.
Nat Commun ; 13(1): 4918, 2022 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-35995799

RESUMO

Considerable evidence supports the release of pathogenic aggregates of the neuronal protein α-Synuclein (αSyn) into the extracellular space. While this release is proposed to instigate the neuron-to-neuron transmission and spread of αSyn pathology in synucleinopathies including Parkinson's disease, the molecular-cellular mechanism(s) remain unclear. To study this, we generated a new mouse model to specifically immunoisolate neuronal lysosomes, and established a long-term culture model where αSyn aggregates are produced within neurons without the addition of exogenous fibrils. We show that neuronally generated pathogenic species of αSyn accumulate within neuronal lysosomes in mouse brains and primary neurons. We then find that neurons release these pathogenic αSyn species via SNARE-dependent lysosomal exocytosis. The released aggregates are non-membrane enveloped and seeding-competent. Additionally, we find that this release is dependent on neuronal activity and cytosolic Ca2+. These results propose lysosomal exocytosis as a central mechanism for the release of aggregated and degradation-resistant proteins from neurons.


Assuntos
Sinucleinopatias , alfa-Sinucleína , Animais , Exocitose , Lisossomos/metabolismo , Camundongos , Neurônios/metabolismo , alfa-Sinucleína/metabolismo
3.
Nat Struct Mol Biol ; 27(2): 192-201, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32042150

RESUMO

Point mutations in cysteine string protein-α (CSPα) cause dominantly inherited adult-onset neuronal ceroid lipofuscinosis (ANCL), a rapidly progressing and lethal neurodegenerative disease with no treatment. ANCL mutations are proposed to trigger CSPα aggregation/oligomerization, but the mechanism of oligomer formation remains unclear. Here we use purified proteins, mouse primary neurons and patient-derived induced neurons to show that the normally palmitoylated cysteine string region of CSPα loses palmitoylation in ANCL mutants. This allows oligomerization of mutant CSPα via ectopic binding of iron-sulfur (Fe-S) clusters. The resulting oligomerization of mutant CSPα causes its mislocalization and consequent loss of its synaptic SNARE-chaperoning function. We then find that pharmacological iron chelation mitigates the oligomerization of mutant CSPα, accompanied by partial rescue of the downstream SNARE defects and the pathological hallmark of lipofuscin accumulation. Thus, the iron chelators deferiprone (L1) and deferoxamine (Dfx), which are already used to treat iron overload in humans, offer a new approach for treating ANCL.


Assuntos
Proteínas de Choque Térmico HSP40/genética , Proteínas de Membrana/genética , Lipofuscinoses Ceroides Neuronais/genética , Mutação Puntual , Agregação Patológica de Proteínas/genética , Animais , Células Cultivadas , Feminino , Células HEK293 , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Quelantes de Ferro/metabolismo , Lipoilação , Proteínas de Membrana/metabolismo , Camundongos , Lipofuscinoses Ceroides Neuronais/metabolismo , Neurônios/metabolismo , Agregação Patológica de Proteínas/metabolismo , Ligação Proteica , Multimerização Proteica
4.
Neurosci Lett ; 705: 183-194, 2019 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-31028844

RESUMO

Alzheimer's disease (AD) is characterized by two major pathological lesions in the brain, amyloid plaques and neurofibrillary tangles (NFTs) composed mainly of amyloid-ß (Aß) peptides and hyperphosphorylated tau, respectively. Although accumulation of toxic Aß species in the brain has been proposed as one of the important early events in AD, continued lack of success of clinical trials based on Aß-targeting drugs has triggered the field to seek out alternative disease mechanisms and related therapeutic strategies. One of the new approaches is to uncover novel roles of pathological tau during disease progression. This review will primarily focus on recent advances in understanding the contributions of tau to AD.


Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Proteínas tau/metabolismo , Proteínas tau/fisiologia , Doença de Alzheimer/complicações , Humanos , Inflamação/complicações , Inflamação/fisiopatologia , Transmissão Sináptica/fisiologia
5.
PLoS One ; 11(9): e0160384, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27611087

RESUMO

Metabolic dysfunction is well-documented in Huntington's disease (HD). However, the link between the mutant huntingtin (mHTT) gene and the pathology is unknown. The tricarboxylic acid (TCA) cycle is the main metabolic pathway for the production of NADH for conversion to ATP via the electron transport chain (ETC). The objective of this study was to test for differences in enzyme activities, mRNAs and protein levels related to the TCA cycle between lymphoblasts from healthy subjects and from patients with HD. The experiments utilize the advantages of lymphoblasts to reveal new insights about HD. The large quantity of homogeneous cell populations permits multiple dynamic measures to be made on exactly comparable tissues. The activities of nine enzymes related to the TCA cycle and the expression of twenty-nine mRNAs encoding for these enzymes and enzyme complexes were measured. Cells were studied under baseline conditions and during metabolic stress. The results support our recent findings that the activities of the pyruvate dehydrogenase complex (PDHC) and succinate dehydrogenase (SDH) are elevated in HD. The data also show a large unexpected depression in MDH activities. Furthermore, message levels for isocitrate dehydrogenase 1 (IDH1) were markedly increased in in HD lymphoblasts and were responsive to treatments. The use of lymphoblasts allowed us to clarify that the reported decrease in aconitase activity in HD autopsy brains is likely due to secondary hypoxic effects. These results demonstrate the mRNA and enzymes of the TCA cycle are critical therapeutic targets that have been understudied in HD.


Assuntos
Ciclo do Ácido Cítrico , Metabolismo Energético , Doença de Huntington/metabolismo , Adulto , Estudos de Casos e Controles , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular , Feminino , Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Doença de Huntington/genética , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Complexo Piruvato Desidrogenase/genética , Complexo Piruvato Desidrogenase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Cianeto de Sódio/farmacologia , Estresse Fisiológico , Repetições de Trinucleotídeos
6.
J Neuropathol Exp Neurol ; 74(6): 527-37, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25978848

RESUMO

Glucose metabolism is reduced in the brains of patients with Huntington disease (HD). The mechanisms underlying this deficit, its link to the pathology of the disease, and the vulnerability of the striatum in HD remain unknown. Abnormalities in some of the key mitochondrial enzymes involved in glucose metabolism, including the pyruvate dehydrogenase complex (PDHC) and the tricarboxylic acid (TCA) cycle, may contribute to these deficits. Here, activities for these enzymes and select protein levels were measured in human postmortem cortex and in striatum and cortex of an HD mouse model (Q175); mRNA levels encoding for these enzymes were also measured in the Q175 mouse cortex. The activities of PDHC and nearly all of the TCA cycle enzymes were dramatically lower (-50% to 90%) in humans than in mice. The activity of succinate dehydrogenase increased with HD in human (35%) and mouse (23%) cortex. No other changes were detected in the human HD cortex or mouse striatum. In Q175 cortex, there were increased activities of PDHC (+12%) and aconitase (+32%). Increased mRNA levels for succinyl thiokinase (+88%) and isocitrate dehydrogenase (+64%) suggested an upregulation of the TCA cycle. These patterns of change differ from those reported in other diseases, which may offer unique metabolic therapeutic opportunities for HD patients.


Assuntos
Córtex Cerebral/enzimologia , Ciclo do Ácido Cítrico/fisiologia , Corpo Estriado/enzimologia , Doença de Huntington , Complexo Cetoglutarato Desidrogenase/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Di-Hidrolipoamida Desidrogenase/genética , Di-Hidrolipoamida Desidrogenase/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Doença de Huntington/enzimologia , Doença de Huntington/patologia , Doença de Huntington/fisiopatologia , Complexo Cetoglutarato Desidrogenase/genética , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação/genética , Mudanças Depois da Morte , Complexo Piruvato Desidrogenase/genética , Complexo Piruvato Desidrogenase/metabolismo , Estudos Retrospectivos
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