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1.
Glia ; 71(8): 1804-1829, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37026600

RESUMO

Autoantibodies against the NR1 subunit of NMDA receptors (NMDARs) have been shown to promote crosslinking and internalization of bound receptors in NMDAR encephalitis (NMDARE). This internalization-mediated loss of NMDARs is thought to be the major mechanism leading to pathogenic outcomes in patients. However, the role of bound autoantibody in engaging the resident immune cells, microglia, remains poorly understood. Here, using a patient-derived monoclonal NR1 autoantibody (hNR1-mAb) and a co-culture system of microglia and neurons, we could show that hNR1-mAb bound to hippocampal neurons led to microglia-mediated removal of hNR1-mAb bound NMDARs. These complexes were found to accumulate inside endo-lysosomal compartments of microglia. Utilizing another patient isolated monoclonal autoantibody, against the α1-subunit of GABAA receptors (α1-GABAA -mAb), such removal of receptors was found to be specific to the antibody-bound receptor targets. Interestingly, along with receptor removal, we also observed a reduction in synapse number, more specifically in the numbers of post-synaptic proteins like PSD95 and Homer 1, when microglia were present in the culture. Importantly, mutations in the Fc region of hNR1-mAb, blocking its Fcγ receptor (FcγR) and complement binding, attenuated hNR1-mAb driven loss of NMDARs and synapses, indicating that microglia engagement by bound hNR1-mAb is critical for receptor and synapse loss. Our data argues for an active involvement of microglia in removal of NMDARs and other receptors in individuals with autoimmune encephalitis, thereby contributing to the etiology of these diseases.


Assuntos
Autoanticorpos , Receptores de N-Metil-D-Aspartato , Humanos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Autoanticorpos/metabolismo , Técnicas de Cocultura , Microglia/metabolismo , Neurônios/metabolismo , Ácido gama-Aminobutírico/metabolismo
2.
Proc Natl Acad Sci U S A ; 117(31): 18661-18669, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32675242

RESUMO

Huntington's disease (HD) is a progressive incurable neurodegenerative disorder characterized by motor and neuropsychiatric symptoms. It is caused by expansion of a cytosine-adenine-guanine triplet in the N-terminal domain of exon 1 in the huntingtin (HTT) gene that codes for an expanded polyglutamine stretch in the protein product which becomes aggregation prone. The mutant Htt (mHtt) aggregates are associated with components of the ubiquitin-proteasome system, suggesting that mHtt is marked for proteasomal degradation and that, for reasons still debated, are not properly degraded. We used a novel HD rat model, proteomic analysis, and long-term live neuronal imaging to characterize the effects of ubiquitination on aggregation of mHtt and subsequent cellular responses. We identified two lysine residues, 6 and 9, in the first exon of mHtt that are specifically ubiquitinated in striatal and cortical brain tissues of mHtt-transgenic animals. Expression of mHtt exon 1 lacking these ubiquitination sites in cortical neurons and cultured cells was found to slow aggregate appearance rates and reduce their size but at the same time increase the number of much smaller and less visible ones. Importantly, expression of this form of mHtt was associated with elevated death rates. Proteomic analysis indicated that cellular reactions to mHtt expression were weaker in cells expressing the lysineless protein, possibly implying a reduced capacity to cope with the proteotoxic stress. Taken together, the findings suggest a novel role for ubiquitination-attenuation of the pathogenic effect of mHtt.


Assuntos
Proteína Huntingtina , Doença de Huntington , Ubiquitinação/fisiologia , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Morte Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Lisina/química , Lisina/metabolismo , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma , Agregação Patológica de Proteínas/metabolismo , Ratos , Ratos Transgênicos
3.
RNA Biol ; 19(1): 333-352, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35220879

RESUMO

Latent 5' splice sites, not normally used, are highly abundant in human introns, but are activated under stress and in cancer, generating thousands of nonsense mRNAs. A previously proposed mechanism to suppress latent splicing was shown to be independent of NMD, with a pivotal role for initiator-tRNA independent of protein translation. To further elucidate this mechanism, we searched for nuclear proteins directly bound to initiator-tRNA. Starting with UV-crosslinking, we identified nucleolin (NCL) interacting directly and specifically with initiator-tRNA in the nucleus, but not in the cytoplasm. Next, we show the association of ini-tRNA and NCL with pre-mRNA. We further show that recovery of suppression of latent splicing by initiator-tRNA complementation is NCL dependent. Finally, upon nucleolin knockdown we show activation of latent splicing in hundreds of coding transcripts having important cellular functions. We thus propose nucleolin, a component of the endogenous spliceosome, through its direct binding to initiator-tRNA and its effect on latent splicing, as the first protein of a nuclear quality control mechanism regulating splice site selection to protect cells from latent splicing that can generate defective mRNAs.


Assuntos
Sítios de Ligação , Fosfoproteínas/metabolismo , Sítios de Splice de RNA , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Espectrometria de Massas , Ligação Proteica , Interferência de RNA , RNA de Transferência/genética , Nucleolina
4.
Nucleic Acids Res ; 46(20): 11014-11029, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30203035

RESUMO

MicroRNAs (miRNAs) are short non-coding RNAs that negatively regulate the expression and translation of genes in healthy and diseased tissues. Herein, we characterize short RNAs from human HeLa cells found in the supraspliceosome, a nuclear dynamic machine in which pre-mRNA processing occurs. We sequenced small RNAs (<200 nt) extracted from the supraspliceosome, and identified sequences that are derived from 200 miRNAs genes. About three quarters of them are mature miRNAs, whereas the rest account for various defined regions of the pre-miRNA, and its hairpin-loop precursor. Out of these aligned sequences, 53 were undetected in cellular extract, and the abundance of additional 48 strongly differed from that in cellular extract. Notably, we describe seven abundant miRNA-derived sequences that overlap non-coding exons of their host gene. The rich collection of sequences identical to pre-miRNAs at the supraspliceosome suggests overlooked nuclear functions. Specifically, the abundant hsa-mir-99b may affect splicing of LINC01129 primary transcript through base-pairing with its exon-intron junction. Using suppression and overexpression experiments, we show that hsa-mir-7704 negatively regulates the level of the lncRNA HAGLR. We claim that in cases of extended base-pairing complementarity, such supraspliceosomal pre-miRNA sequences might have a role in transcription attenuation, maturation and processing.


Assuntos
MicroRNAs/genética , Precursores de RNA/genética , Spliceossomos/genética , Sequência de Bases , Linhagem Celular , Regulação da Expressão Gênica , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Splicing de RNA , Spliceossomos/metabolismo
5.
Commun Biol ; 7(1): 1014, 2024 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-39155290

RESUMO

Huntington's disease (HD) is caused by a glutamine repeat expansion in the protein huntingtin. Mutated huntingtin (mHtt) forms aggregates whose impacts on neuronal survival are still debated. Using weeks-long, continual imaging of cortical neurons, we find that mHtt is gradually sequestrated into peripheral, mainly axonal aggregates, concomitant with dramatic reductions in cytosolic mHtt levels and enhanced neuronal survival. in-situ pulse-chase imaging reveals that aggregates continually gain and lose mHtt, in line with these acting as mHtt sinks at equilibrium with cytosolic pools. Mutating two N-terminal lysines found to be ubiquitinated in HD animal models suppresses peripheral aggregate formation and reductions in cytosolic mHtt, promotes nuclear aggregate formation, stabilizes aggregates and leads to pervasive neuronal death. These findings demonstrate the capacity of aggregates formed at peripheral locations to sequester away cytosolic, presumably toxic mHtt forms and support a crucial role for N-terminal ubiquitination in promoting these processes and delaying neuronal death.


Assuntos
Morte Celular , Proteína Huntingtina , Doença de Huntington , Neurônios , Ubiquitinação , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Animais , Neurônios/metabolismo , Neurônios/patologia , Doença de Huntington/metabolismo , Doença de Huntington/genética , Doença de Huntington/patologia , Camundongos , Humanos
6.
F1000Res ; 92020.
Artigo em Inglês | MEDLINE | ID: mdl-32518633

RESUMO

Background: HaloTag is a modified bacterial enzyme that binds rapidly and irreversibly to an array of synthetic ligands, including chemical dyes. When expressed in live cells in conjunction with a protein of interest, HaloTag can be used to study protein trafficking, synthesis, and degradation. For instance, sequential HaloTag labeling with spectrally separable dyes can be used to separate preexisting protein pools from proteins newly synthesized following experimental manipulations or the passage of time. Unfortunately, incomplete labeling by the first dye, or labeling by residual, trapped dye pools can confound interpretation. Methods: Labeling specificity of newly synthesized proteins could be improved by blocking residual binding sites. To that end, we synthesized a non-fluorescent, cell permeable blocker (1-chloro-6-(2-propoxyethoxy)hexane; CPXH), essentially the HaloTag ligand backbone without the reactive amine used to attach fluorescent groups. Results: High-content imaging was used to quantify the ability of CPXH to block HaloTag ligand binding in live HEK cells expressing a fusion protein of mTurquoise2 and HaloTag. Full saturation was observed at CPXH concentrations of 5-10 µM at 30 min. No overt effects on cell viability were observed at any concentration or treatment duration. The ability of CPXH to improve the reliability of newly synthesized protein detection was then demonstrated in live cortical neurons expressing the mTurquoise2-HaloTag fusion protein, in both single and dual labeling time lapse experiments. Practically no labeling was observed after blocking HaloTag binding sites with CPXH when protein synthesis was suppressed with cycloheximide, confirming the identification of newly synthesized protein copies as such, while providing estimates of protein synthesis suppression in these experiments. Conclusions: CPXH is a reliable (and inexpensive) non-fluorescent ligand for improving assessment of protein-of-interest metabolism in live cells using HaloTag technology.


Assuntos
Corantes , Biossíntese de Proteínas , Proteínas , Animais , Células Cultivadas , Feminino , Células HEK293 , Humanos , Ligantes , Masculino , Neurônios , Transporte Proteico , Ratos , Ratos Wistar , Reprodutibilidade dos Testes
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