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1.
Neuron ; 112(9): 1397-1415.e6, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38377989

RESUMO

Defects in tRNA biogenesis are associated with multiple neurological disorders, yet our understanding of these diseases has been hampered by an inability to determine tRNA expression in individual cell types within a complex tissue. Here, we developed a mouse model in which RNA polymerase III is conditionally epitope tagged in a Cre-dependent manner, allowing us to accurately profile tRNA expression in any cell type in vivo. We investigated tRNA expression in diverse nervous system cell types, revealing dramatic heterogeneity in the expression of tRNA genes between populations. We found that while maintenance of levels of tRNA isoacceptor families is critical for cellular homeostasis, neurons are differentially vulnerable to insults to distinct tRNA isoacceptor families. Cell-type-specific translatome analysis suggests that the balance between tRNA availability and codon demand may underlie such differential resilience. Our work provides a platform for investigating the complexities of mRNA translation and tRNA biology in the brain.


Assuntos
Encéfalo , Homeostase , Neurônios , RNA de Transferência , Animais , RNA de Transferência/genética , RNA de Transferência/metabolismo , Homeostase/fisiologia , Camundongos , Encéfalo/metabolismo , Neurônios/metabolismo , RNA Polimerase III/metabolismo , RNA Polimerase III/genética , Camundongos Transgênicos
2.
Biology (Basel) ; 12(2)2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36829435

RESUMO

Boosting trophic support to striatal neurons by increasing levels of brain-derived neurotrophic factor (BDNF) has been considered as a target for therapeutic intervention for several neurodegenerative diseases, including Huntington's disease (HD). To aid in the implementation of such a strategy, a thorough understanding of BDNF cortical-striatal transport is critical to help guide its strategic delivery. In this manuscript, we investigate the dynamic behavior of BDNF transport along the cortical-striatal axis in Q140 primary neurons, a mouse model for HD. We examine this by using single-molecule labeling of BDNF conjugated with quantum dots (QD-BDNF) to follow the transport along the cortical-striatal axis in a microfluidic chamber system specifically designed for the co-culture of cortical and striatal primary neurons. Using this approach, we observe a defect of QD-BDNF transport in Q140 neurons. Our study demonstrates that QD-BDNF transport along the cortical-striatal axis involves the impairment of anterograde transport within axons of cortical neurons, and of retrograde transport within dendrites of striatal neurons. One prominent feature we observe is the extended pause time of QD-BDNF retrograde transport within Q140 striatal dendrites. Taken together, these finding support the hypothesis that delinquent spatiotemporal trophic support of BDNF to striatal neurons, driven by impaired transport, may contribute to the pathogenesis of HD, providing us with insight into how a BDNF supplementation therapeutic strategy may best be applied for HD.

3.
Proc Natl Acad Sci U S A ; 119(10): e2119529119, 2022 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-35238631

RESUMO

SignificanceUnderstanding and treating neurological disorders are global priorities. Some of these diseases are engendered by mutations that cause defects in the cellular synthesis of transfer RNAs (tRNAs), which function as adapter molecules that translate messenger RNAs into proteins. During tRNA biogenesis, ribonuclease P catalyzes removal of the transcribed sequence upstream of the mature tRNA. Here, we focus on a cytoplasmic tRNAArgUCU that is expressed specifically in neurons and, when harboring a particular point mutation, contributes to neurodegeneration in mice. Our results suggest that this mutation favors stable alternative structures that are not cleaved by mouse ribonuclease P and motivate a paradigm that may help to understand the molecular basis for disease-associated mutations in other tRNAs.


Assuntos
Homeostase , Neurônios/metabolismo , Conformação de Ácido Nucleico , RNA de Transferência/metabolismo , Animais , Pareamento de Bases , Córtex Cerebral/enzimologia , Magnésio/metabolismo , Camundongos , Modelos Moleculares , Mutação Puntual , Processamento de Proteína Pós-Traducional , RNA de Transferência/química , RNA de Transferência/genética , Ribonuclease P/isolamento & purificação , Ribonuclease P/metabolismo , Especificidade por Substrato
4.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34548404

RESUMO

Homozygous mutation of the RNA kinase CLP1 (cleavage factor polyribonucleotide kinase subunit 1) causes pontocerebellar hypoplasia type 10 (PCH10), a pediatric neurodegenerative disease. CLP1 is associated with the transfer RNA (tRNA) splicing endonuclease complex and the cleavage and polyadenylation machinery, but its function remains unclear. We generated two mouse models of PCH10: one homozygous for the disease-associated Clp1 mutation, R140H, and one heterozygous for this mutation and a null allele. Both models exhibit loss of lower motor neurons and neurons of the deep cerebellar nuclei. To explore whether Clp1 mutation impacts tRNA splicing, we profiled the products of intron-containing tRNA genes. While mature tRNAs were expressed at normal levels in mutant mice, numerous other products of intron-containing tRNA genes were dysregulated, with pre-tRNAs, introns, and certain tRNA fragments up-regulated, and other fragments down-regulated. However, the spatiotemporal patterns of dysregulation do not correlate with pathogenicity for most altered tRNA products. To elucidate the effect of Clp1 mutation on precursor messenger RNA (pre-mRNA) cleavage, we analyzed poly(A) site (PAS) usage and gene expression in Clp1R140H/- spinal cord. PAS usage was shifted from proximal to distal sites in the mutant mouse, particularly in short and closely spaced genes. Many such genes were also expressed at lower levels in the Clp1R140H/- mouse, possibly as a result of impaired transcript maturation. These findings are consistent with the hypothesis that select genes are particularly dependent upon CLP1 for proper pre-mRNA cleavage, suggesting that impaired mRNA 3' processing may contribute to pathogenesis in PCH10.


Assuntos
Doenças Cerebelares/patologia , Doenças Neurodegenerativas/patologia , Poliadenilação , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/fisiologia , Fatores de Transcrição/fisiologia , Animais , Doenças Cerebelares/genética , Doenças Cerebelares/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA de Transferência/genética
5.
Nucleic Acids Res ; 49(7): 3603-3616, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33341895

RESUMO

During mRNA translation, tRNAs are charged by aminoacyl-tRNA synthetases and subsequently used by ribosomes. A multi-enzyme aminoacyl-tRNA synthetase complex (MSC) has been proposed to increase protein synthesis efficiency by passing charged tRNAs to ribosomes. An alternative function is that the MSC repurposes specific synthetases that are released from the MSC upon cues for functions independent of translation. To explore this, we generated mammalian cells in which arginyl-tRNA synthetase and/or glutaminyl-tRNA synthetase were absent from the MSC. Protein synthesis, under a variety of stress conditions, was unchanged. Most strikingly, levels of charged tRNAArg and tRNAGln remained unchanged and no ribosome pausing was observed at codons for arginine and glutamine. Thus, increasing or regulating protein synthesis efficiency is not dependent on arginyl-tRNA synthetase and glutaminyl-tRNA synthetase in the MSC. Alternatively, and consistent with previously reported ex-translational roles requiring changes in synthetase cellular localizations, our manipulations of the MSC visibly changed localization.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Biossíntese de Proteínas , RNA de Transferência de Arginina/metabolismo , RNA de Transferência de Glutamina/metabolismo , Ribossomos/metabolismo , Animais , Fibroblastos , Células HEK293 , Humanos , Camundongos
6.
Neuron ; 108(1): 193-208.e9, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-32853550

RESUMO

The mammalian genome has hundreds of nuclear-encoded tRNAs, but the contribution of individual tRNA genes to cellular and organismal function remains unknown. Here, we demonstrate that mutations in a neuronally enriched arginine tRNA, n-Tr20, increased seizure threshold and altered synaptic transmission. n-Tr20 expression also modulated seizures caused by an epilepsy-linked mutation in Gabrg2, a gene encoding a GABAA receptor subunit. Loss of n-Tr20 altered translation initiation by activating the integrated stress response and suppressing mTOR signaling, the latter of which may contribute to altered neurotransmission in mutant mice. Deletion of a highly expressed isoleucine tRNA similarly altered these signaling pathways in the brain, suggesting that regulation of translation initiation is a conserved response to tRNA loss. Our data indicate that loss of a single member of a tRNA family results in multiple cellular phenotypes, highlighting the disease-causing potential of tRNA mutations.


Assuntos
Neurônios/metabolismo , RNA de Transferência de Arginina/genética , Convulsões/genética , Transmissão Sináptica/genética , Animais , Eletrochoque/efeitos adversos , Antagonistas de Receptores de GABA-A/efeitos adversos , Camundongos , Pentilenotetrazol/efeitos adversos , Iniciação Traducional da Cadeia Peptídica/genética , RNA de Transferência de Isoleucina/genética , RNA-Seq , Receptores de GABA-A/genética , Convulsões/induzido quimicamente , Convulsões/etiologia , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
7.
Trends Genet ; 34(3): 218-231, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29352613

RESUMO

Errors during mRNA translation can lead to a reduction in the levels of functional proteins and an increase in deleterious molecules. Advances in next-generation sequencing have led to the discovery of rare genetic disorders, many caused by mutations in genes encoding the mRNA translation machinery, as well as to a better understanding of translational dynamics through ribosome profiling. We discuss here multiple neurological disorders that are linked to errors in tRNA aminoacylation and ribosome decoding. We draw on studies from genetic models, including yeast and mice, to enhance our understanding of the translational defects observed in these diseases. Finally, we emphasize the importance of tRNA, their associated enzymes, and the inextricable link between accuracy and efficiency in the maintenance of translational fidelity.


Assuntos
Mutação , Doenças do Sistema Nervoso/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Animais , Humanos , Modelos Genéticos , Saccharomyces cerevisiae/genética , Aminoacilação de RNA de Transferência/genética
8.
Neuron ; 96(3): 616-637, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-29096076

RESUMO

Dynamic regulation of mRNA translation initiation and elongation is essential for the survival and function of neural cells. Global reductions in translation initiation resulting from mutations in the translational machinery or inappropriate activation of the integrated stress response may contribute to pathogenesis in a subset of neurodegenerative disorders. Aberrant proteins generated by non-canonical translation initiation may be a factor in the neuron death observed in the nucleotide repeat expansion diseases. Dysfunction of central components of the elongation machinery, such as the tRNAs and their associated enzymes, can cause translational infidelity and ribosome stalling, resulting in neurodegeneration. Taken together, dysregulation of mRNA translation is emerging as a unifying mechanism underlying the pathogenesis of many neurodegenerative disorders.


Assuntos
Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Neurônios/fisiologia , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/fisiologia , Animais , Morte Celular/fisiologia , Citoplasma/genética , Citoplasma/metabolismo , Humanos
9.
Cell Mol Life Sci ; 71(20): 4043-54, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24687423

RESUMO

The retrograde transport of endosomes within axons proceeds with remarkable uniformity despite having to navigate a discontinuous microtubule network. The mechanisms through which this navigation is achieved remain elusive. In this report, we demonstrate that access of SxIP motif proteins, such as BPAG1n4, to the microtubule plus end is important for the maintenance of processive and sustained retrograde transport along the axon. Disruption of this interaction at the microtubule plus end significantly increases endosome stalling. Our study thus provides strong insight into the role of plus-end-binding proteins in the processive navigation of cargo within the axon.


Assuntos
Axônios/metabolismo , Proteínas do Citoesqueleto/química , Microtúbulos/metabolismo , Motivos de Aminoácidos , Animais , Transporte Axonal , Células COS , Células Cultivadas , Chlorocebus aethiops , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Endossomos/metabolismo , Células HEK293 , Humanos , Microtúbulos/química , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
10.
J Biol Chem ; 287(49): 41139-51, 2012 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-23060447

RESUMO

Mutations in the P4-ATPase ATP8B1 cause the inherited liver disease progressive familial intrahepatic cholestasis. Several of these mutations are located in conserved regions of the transmembrane domain associated with substrate binding and transport. Assays for P4-ATPase-mediated transport in living yeast cells were developed and used to characterize the specificity and kinetic parameters of this transport. Progressive familial intrahepatic cholestasis mutations were introduced into the yeast plasma membrane P4-ATPase Dnf2p, and the effect of these mutations on its catalysis of phospholipid transport were determined. The results of these measurements have implications for the basis of the disease and for the mechanism of phospholipid transit through the enzyme during the reaction cycle.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Colestase Intra-Hepática/genética , Mutação , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Humanos , Cinética , Dados de Sequência Molecular , Fenótipo , Fosfolipídeos/química , Análise de Regressão , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
11.
EMBO Rep ; 13(11): 1021-9, 2012 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-22995871

RESUMO

Microtubules (MTs) are integral to numerous cellular functions, such as cell adhesion, differentiation and intracellular transport. Their dynamics are largely controlled by diverse MT-interacting proteins, but the signalling mechanisms that regulate these interactions remain elusive. In this report, we identify a rapid, calcium-regulated switch between MT plus end interaction and lattice binding within the carboxyl terminus of BPAG1n4. This switch is EF-hand dependent, and mutations of the EF-hands abolish this dynamic behaviour. Our study thus uncovers a new, calcium-dependent regulatory mechanism for a spectraplakin, BPAG1n4, at the MT plus end.


Assuntos
Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Células COS , Proteínas de Transporte/química , Proteínas de Transporte/genética , Chlorocebus aethiops , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Distonina , Motivos EF Hand , Células HEK293 , Humanos , Microtúbulos/química , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética
12.
PLoS One ; 7(4): e33094, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22523538

RESUMO

In neurons, a highly regulated microtubule cytoskeleton is essential for many cellular functions. These include axonal transport, regional specialization and synaptic function. Given the critical roles of microtubule-associated proteins (MAPs) in maintaining and regulating microtubule stability and dynamics, we sought to understand how this regulation is achieved. Here, we identify a novel LisH/WD40 repeat protein, tentatively named nemitin (neuronal enriched MAP interacting protein), as a potential regulator of MAP8-associated microtubule function. Based on expression at both the mRNA and protein levels, nemitin is enriched in the nervous system. Its protein expression is detected as early as embryonic day 11 and continues through adulthood. Interestingly, when expressed in non-neuronal cells, nemitin displays a diffuse pattern with puncta, although at the ultrastructural level it localizes along the microtubule network in vivo in sciatic nerves. These results suggest that the association of nemitin to microtubules may require an intermediary protein. Indeed, co-expression of nemitin with microtubule-associated protein 8 (MAP8) results in nemitin losing its diffuse pattern, instead decorating microtubules uniformly along with MAP8. Together, these results imply that nemitin may play an important role in regulating the neuronal cytoskeleton through an interaction with MAP8.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/metabolismo , Sequência de Aminoácidos , Animais , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Proteínas dos Microfilamentos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/efeitos dos fármacos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso , Neurônios/metabolismo
13.
PLoS One ; 5(6): e11151, 2010 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-20567601

RESUMO

Lithium (Li(+)) is a common treatment for bipolar mood disorder, a major psychiatric illness with a lifetime prevalence of more than 1%. Risk of bipolar disorder is heavily influenced by genetic predisposition, but is a complex genetic trait and, to date, genetic studies have provided little insight into its molecular origins. An alternative approach is to investigate the genetics of Li(+) sensitivity. Using the social amoeba Dictyostelium, we previously identified prolyl oligopeptidase (PO) as a modulator of Li(+) sensitivity. In a link to the clinic, PO enzyme activity is altered in bipolar disorder patients. Further studies demonstrated that PO is a negative regulator of inositol(1,4,5)trisphosphate (IP(3)) synthesis, a Li(+) sensitive intracellular signal. However, it was unclear how PO could influence either Li(+) sensitivity or risk of bipolar disorder. Here we show that in both Dictyostelium and cultured human cells PO acts via Multiple Inositol Polyphosphate Phosphatase (Mipp1) to control gene expression. This reveals a novel, gene regulatory network that modulates inositol metabolism and Li(+) sensitivity. Among its targets is the inositol monophosphatase gene IMPA2, which has also been associated with risk of bipolar disorder in some family studies, and our observations offer a cellular signalling pathway in which PO activity and IMPA2 gene expression converge.


Assuntos
Resistência a Medicamentos/genética , Regulação da Expressão Gênica , Inositol/biossíntese , Compostos de Lítio/farmacologia , Quimiotaxia/efeitos dos fármacos , Dictyostelium/genética , Monoéster Fosfórico Hidrolases/metabolismo
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