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1.
Stem Cells ; 42(4): 317-328, 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38227647

RESUMO

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have unique characteristics where they can both contribute to all three germ layers in vivo and self-renewal indefinitely in vitro. Post-translational modifications of proteins, particularly by the ubiquitin proteasome system (UPS), control cell pluripotency, self-renewal, and differentiation. A significant number of UPS members (mainly ubiquitin ligases) regulate pluripotency and influence ESC differentiation with key elements of the ESC pluripotency network (including the "master" regulators NANOG and OCT4) being controlled by ubiquitination. To further understand the role of the UPS in pluripotency, we performed an RNAi screen during induction of cellular reprogramming and have identified FBXO9 as a novel regulator of pluripotency associated protein DPPA5. Our findings indicate that FBXO9 silencing facilitates the induction of pluripotency through decreased proteasomal degradation of DPPA5. These findings identify FBXO9 as a key regulator of pluripotency.


Assuntos
Células-Tronco Embrionárias , Proteínas F-Box , Complexo de Endopeptidases do Proteassoma , Ubiquitina-Proteína Ligases , Diferenciação Celular , Reprogramação Celular , Células-Tronco Embrionárias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Camundongos , Proteínas F-Box/genética , Proteínas F-Box/metabolismo
2.
Leukemia ; 37(11): 2197-2208, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37689825

RESUMO

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by clonal expansion of myeloid blasts in the bone marrow (BM). Despite advances in therapy, the prognosis for AML patients remains poor, and there is a need to identify novel molecular pathways regulating tumor cell survival and proliferation. F-box ubiquitin E3 ligase, FBXO21, has low expression in AML, but expression correlates with survival in AML patients and patients with higher expression have poorer outcomes. Silencing FBXO21 in human-derived AML cell lines and primary patient samples leads to differentiation, inhibition of tumor progression, and sensitization to chemotherapy agents. Additionally, knockdown of FBXO21 leads to up-regulation of cytokine signaling pathways. Through a mass spectrometry-based proteomic analysis of FBXO21 in AML, we identified that FBXO21 ubiquitylates p85α, a regulatory subunit of the phosphoinositide 3-kinase (PI3K) pathway, for degradation resulting in decreased PI3K signaling, dimerization of free p85α and ERK activation. These findings reveal the ubiquitin E3 ligase, FBXO21, plays a critical role in regulating AML pathogenesis, specifically through alterations in PI3K via regulation of p85α protein stability.


Assuntos
Proteínas F-Box , Leucemia Mieloide Aguda , Humanos , Proliferação de Células/fisiologia , Proteínas F-Box/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Proteômica , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismo
3.
Exp Hematol ; 114: 33-42.e3, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35987460

RESUMO

Hematopoietic cell fate decisions such as self-renewal and differentiation are highly regulated through multiple molecular pathways. One pathway, the ubiquitin proteasome system (UPS), controls protein levels by tagging them with polyubiquitin chains and promoting their degradation through the proteasome. Ubiquitin E3 ligases serve as the substrate-recognition component of the UPS. By investigating the FBOX family of E3 ligases, we discovered that Fbxo21 was highly expressed in the hematopoietic stem and progenitor cell (HSPC) population, and exhibited low to no expression in mature myeloid populations. To determine the role of FBXO21 on HSPC maintenance, self-renewal, and differentiation, we generated shRNAs against FBXO21 and a hematopoiesis-specific Fbxo21 conditional knockout (cKO) mouse model. We found that silencing FBXO21 in HSPCs led to a loss in colony formation and an increase in cell differentiation in vitro. Additionally, stressing the HSPC populations in our Fbxo21 cKO mouse with 5-fluorouracil injections resulted in a decrease in survival, despite these populations exhibiting minimal alterations during steady-state hematopoiesis. Although FBXO21 has previously been proposed to regulate cytokine signaling via ASK and p38, our results indicate that depletion of FBXO21 led to altered ERK signaling in vitro. Together, these findings suggest ubiquitin E3 ligase FBXO21 regulates HSPCs through cytokine-mediated pathways.


Assuntos
Citocinas , Proteínas F-Box , Transdução de Sinais , Ubiquitina-Proteína Ligases , Animais , Citocinas/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Fluoruracila , Hematopoese , Camundongos , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
4.
Leukemia ; 36(5): 1296-1305, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35177813

RESUMO

Acute myeloid leukemia (AML) is a devastating cancer affecting the hematopoietic system. Previous research has relied on RNA sequencing and microarray techniques to study the downstream effects of genomic alterations. While these studies have proven efficacious, they fail to capture the changes that occur at the proteomic level. To interrogate the effect of protein expression alterations in AML, we performed a quantitative mass spectrometry in parallel with RNAseq analysis using AML mouse models. These combined results identified 34 proteins whose expression was upregulated in AML tumors, but strikingly, were unaltered at the transcriptional level. Here we focus on mitochondrial electron transfer proteins ETFA and ETFB. Silencing of ETFA and ETFB led to increased mitochondrial activity, mitochondrial stress, and apoptosis in AML cells, but had little to no effect on normal human CD34+ cells. These studies identify a set of proteins that have not previously been associated with leukemia and may ultimately serve as potential targets for therapeutic manipulation to hinder AML progression and help contribute to our understanding of the disease.


Assuntos
Leucemia Mieloide Aguda , Proteínas Mitocondriais , Animais , Apoptose , Descoberta de Drogas , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/farmacologia , Proteômica
5.
Haematologica ; 107(3): 690-701, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33792219

RESUMO

B-cell non-Hodgkin lymphoma (B-NHL) encompasses multiple clinically and phenotypically distinct subtypes of malignancy with unique molecular etiologies. Common subtypes of B-NHL, such as diffuse large B-cell lymphoma, have been comprehensively interrogated at the genomic level, but rarer subtypes, such as mantle cell lymphoma, remain less extensively characterized. Furthermore, multiple B-NHL subtypes have thus far not been comprehensively compared using the same methodology to identify conserved or subtype-specific patterns of genomic alterations. Here, we employed a large targeted hybrid-capture sequencing approach encompassing 380 genes to interrogate the genomic landscapes of 685 B-NHL tumors at high depth, including diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, and Burkitt lymphoma. We identified conserved hallmarks of B-NHL that were deregulated in the majority of tumors from each subtype, including frequent genetic deregulation of the ubiquitin proteasome system. In addition, we identified subtype-specific patterns of genetic alterations, including clusters of co-occurring mutations and DNA copy number alterations. The cumulative burden of mutations within a single cluster were more discriminatory of B-NHL subtypes than individual mutations, implicating likely patterns of genetic cooperation that contribute to disease etiology. We therefore provide the first cross-sectional analysis of mutations and DNA copy number alterations across major B-NHL subtypes and a framework of co-occurring genetic alterations that deregulate genetic hallmarks and likely cooperate in lymphomagenesis.


Assuntos
Linfoma de Burkitt , Linfoma Folicular , Linfoma Difuso de Grandes Células B , Adulto , Estudos Transversais , Humanos , Linfoma Folicular/genética , Mutação
6.
Biol Open ; 10(1)2021 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-33408128

RESUMO

RNA polymerase II subunit A Carboxy-Terminal Domain Phosphatase 1 (CTDP1), a member of the haloacid dehalogenase superfamily phosphatases, has a defined role in transcriptional regulation, but emerging evidence suggests an expanded functional repertoire in the cell cycle and DNA damage response. In humans, a splice site mutation in CTDP1 gives rise to the rare Congenital Cataracts Facial Dysmorphism and Neuropathy syndrome, and recent evidence from our lab indicates CTDP1 is required for breast cancer growth and proliferation. To explore the physiological function of CTDP1 in a mammalian system, we generated a conditional Ctdp1 knockout mouse model by insertion of loxP sites upstream of exon 3 and downstream of exon 4. Biallelic deletion of Ctdp1 results in lethality before embryonic day 7.5, with morphological features indicating embryo cell death and resorption. However, Ctdp1+/- mice are haplosufficient for phenotypic traits including body weight, hematological parameters, exploratory and locomotive functions. To investigate the potential mechanisms of the embryonic death caused by biallelic Ctdp1 knockout, mouse embryonic fibroblasts (MEFs) were established from Ctdp1+/+ and Ctdp1flox/flox mice. Lentivirus delivered Cre-mediated biallelic deletion of Ctdp1 in MEFs results in cell death preceded by impaired proliferation characterized by an increase in G1- and G2-phase populations and a reduction in the S-phase population. These cell cycle alterations caused by deletion of Ctdp1 are associated with an increase in p27 protein expression and a decrease in phosphorylated RB, phosphorylated Histone H3, and Cyclin B expression. Together, these results reveal that Ctdp1 plays an essential role in early mouse embryo development and cell growth and survival in part by regulating the cell cycle.


Assuntos
Ciclo Celular/genética , Desenvolvimento Embrionário/genética , Fibroblastos/metabolismo , Genes Letais , Fosfoproteínas Fosfatases/deficiência , Animais , Pontos de Checagem do Ciclo Celular/genética , Morte Celular/genética , Linhagem Celular , Deleção de Genes , Marcação de Genes , Vetores Genéticos/genética , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Fenótipo
7.
Trends Immunol ; 41(12): 1128-1140, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33160841

RESUMO

Hematopoiesis is responsible for numerous functions, ranging from oxygen transportation to host defense, to injury repair. This process of hematopoiesis is maintained throughout life by hematopoietic stem cells and requires a controlled balance between self-renewal, differentiation, and quiescence. Disrupting this balance can result in hematopoietic malignancies, including anemia, immune deficiency, leukemia, and lymphoma. Recent work has shown that FBOX E3 ligases, a substrate recognition component of the ubiquitin proteasome system (UPS), have an integral role in maintaining this balance. In this review, we detail how FBOX proteins target specific proteins for degradation to regulate hematopoiesis through cell processes, such as cell cycle, development, and apoptosis.


Assuntos
Hematopoese , Proteólise , Ubiquitina-Proteína Ligases , Proteínas F-Box/metabolismo , Hematopoese/genética , Células-Tronco Hematopoéticas , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
8.
Blood ; 136(3): 299-312, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32325489

RESUMO

Coordination of a number of molecular mechanisms including transcription, alternative splicing, and class switch recombination are required to facilitate development, activation, and survival of B cells. Disruption of these pathways can result in malignant transformation. Recently, next-generation sequencing has identified a number of novel mutations in mantle cell lymphoma (MCL) patients including mutations in the ubiquitin E3 ligase UBR5. Approximately 18% of MCL patients were found to have mutations in UBR5, with the majority of mutations within the HECT domain of the protein that can accept and transfer ubiquitin molecules to the substrate. Determining if UBR5 controls the maturation of B cells is important to fully understand malignant transformation to MCL. To elucidate the role of UBR5 in B-cell maturation and activation, we generated a conditional mutant disrupting UBR5's C-terminal HECT domain. Loss of the UBR5 HECT domain leads to a block in maturation of B cells in the spleen and upregulation of proteins associated with messenger RNA splicing via the spliceosome. Our studies reveal a novel role of UBR5 in B-cell maturation by stabilization of spliceosome components during B-cell development and suggests UBR5 mutations play a role in MCL transformation.


Assuntos
Linfócitos B/enzimologia , Linfoma de Célula do Manto/enzimologia , Mutação , Proteínas de Neoplasias/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Humanos , Linfoma de Célula do Manto/genética , Camundongos , Camundongos Mutantes , Proteínas de Neoplasias/genética , Domínios Proteicos , Ubiquitina-Proteína Ligases/genética
9.
Cancers (Basel) ; 11(11)2019 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-31684170

RESUMO

The hematopoietic system is maintained throughout life by stem cells that are capable of differentiating into all hematopoietic lineages. An intimate balance between self-renewal, differentiation, and quiescence is required to maintain hematopoiesis and disruption of this balance can result in malignant transformation. FBXO9, the substrate recognition component from the SCF E3 ubiquitin ligase family, is downregulated in patients with acute myeloid leukemia (AML) compared to healthy bone marrow, and this downregulation is particularly evident in patients with inv(16) AML. To study FBXO9 in malignant hematopoiesis, we generated a conditional knockout mouse model using a novel CRISPR/Cas9 strategy. Deletion of Fbxo9 in the murine hematopoietic system showed no adverse effects on stem and progenitor cell function but in AML lead to markedly accelerated and aggressive leukemia development in mice with inv(16). Not only did Fbxo9 play a role in leukemia initiation but it also functioned to maintain AML activity and promote disease progression. Quantitative mass spectrometry from primary tumors reveals tumors lacking Fbxo9 highly express proteins associated with metastasis and invasion as well as components of the ubiquitin proteasome system. We confirmed that the loss of FBXO9 leads to increased proteasome activity and tumors cells were more sensitive to in vitro proteasome inhibition with bortezomib, suggesting that FBXO9 expression may predict patients' response to bortezomib.

10.
Neurobiol Aging ; 82: 110-119, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31437719

RESUMO

The goal of this study was to compare regional brain atrophy patterns in cognitively unimpaired (CU) older adults with and without brain accumulation of amyloid-ß (Aß) to elucidate contributions of Aß, age, and other variables to atrophy rates. In 80 CU participants from the Alzheimer's Disease Neuroimaging Initiative, we determined effects of Aß and age on longitudinal, regional atrophy rates, while accounting for confounding variables including sex, APOE ε4 genotype, white matter lesions, and cerebrospinal fluid total and phosphorylated tau levels. We not only found overlapping patterns of atrophy in Aß+ versus Aß- participants but also identified regions where atrophy pattern differed between the 2 groups. Higher Aß load was associated with increased longitudinal atrophy in the entorhinal cortex, amygdala, and hippocampus, even when accounting for age and other variables. Age was associated with atrophy in insula, fusiform gyrus, and isthmus cingulate, even when accounting for Aß. We found age by Aß interactions in the postcentral gyrus and lateral orbitofrontal cortex. These results elucidate the separate and related effects of age, Aß, and other important variables on longitudinal brain atrophy rates in CU older adults.


Assuntos
Envelhecimento/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Cognição/fisiologia , Idoso , Idoso de 80 Anos ou mais , Atrofia , Disfunção Cognitiva/diagnóstico por imagem , Disfunção Cognitiva/metabolismo , Bases de Dados Factuais/tendências , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade
11.
Genome Biol ; 18(1): 92, 2017 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-28511701

RESUMO

BACKGROUND: Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient. RESULTS: Here we describe Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that Easi-CRISPR generates correctly targeted conditional and insertion alleles in 8.5-100% of the resulting live offspring. CONCLUSIONS: Easi-CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus, Easi-CRISPR offers a comprehensive means of building large-scale Cre-LoxP animal resources.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , Camundongos Transgênicos/genética , Mutagênese Insercional/métodos , Ribonucleoproteínas/genética , Animais , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Efeito Fundador , Genes Reporter , Loci Gênicos , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Transgênicos/crescimento & desenvolvimento , Microinjeções , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Reparo de DNA por Recombinação , Ribonucleoproteínas/metabolismo , Zigoto/crescimento & desenvolvimento , Zigoto/metabolismo
12.
Nat Cell Biol ; 18(11): 1127-1138, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27749823

RESUMO

Pluripotent embryonic stem cells (ESCs) self-renew or differentiate into all tissues of the developing embryo and cell-specification factors are necessary to balance gene expression. Here we delineate the function of the PHD-finger protein 5a (Phf5a) in ESC self-renewal and ascribe its role in regulating pluripotency, cellular reprogramming and myoblast specification. We demonstrate that Phf5a is essential for maintaining pluripotency, since depleted ESCs exhibit hallmarks of differentiation. Mechanistically, we attribute Phf5a function to the stabilization of the Paf1 transcriptional complex and control of RNA polymerase II elongation on pluripotency loci. Apart from an ESC-specific factor, we demonstrate that Phf5a controls differentiation of adult myoblasts. Our findings suggest a potent mode of regulation by Phf5a in stem cells, which directs their transcriptional programme, ultimately regulating maintenance of pluripotency and cellular reprogramming.


Assuntos
Proteínas de Transporte/genética , Diferenciação Celular/genética , Reprogramação Celular/genética , Células-Tronco Embrionárias Murinas/citologia , Mioblastos/citologia , Células-Tronco Pluripotentes/citologia , Transcrição Gênica , Envelhecimento , Animais , Linhagem Celular , Proliferação de Células/genética , Proteínas de Ligação a DNA , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a RNA , Transativadores
13.
Elife ; 42015 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-26613412

RESUMO

Little is known on post-transcriptional regulation of adult and embryonic stem cell maintenance and differentiation. Here we characterize the role of Ddb1, a component of the CUL4-DDB1 ubiquitin ligase complex. Ddb1 is highly expressed in multipotent hematopoietic progenitors and its deletion leads to abrogation of both adult and fetal hematopoiesis, targeting specifically transiently amplifying progenitor subsets. However, Ddb1 deletion in non-dividing lymphocytes has no discernible phenotypes. Ddb1 silencing activates Trp53 pathway and leads to significant effects on cell cycle progression and rapid apoptosis. The abrogation of hematopoietic progenitor cells can be partially rescued by simultaneous deletion of Trp53. Conversely, depletion of DDB1 in embryonic stem cell (ESC) leads to differentiation albeit negative effects on cell cycle and apoptosis. Mass spectrometry reveals differing protein interactions between DDB1 and distinct DCAFs, the substrate recognizing components of the E3 complex, between cell types. Our studies identify CUL4-DDB1 complex as a novel post-translational regulator of stem and progenitor maintenance and differentiation.


Assuntos
Diferenciação Celular , Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/fisiologia , Animais , Proteínas de Ligação a DNA/genética , Deleção de Genes , Regulação da Expressão Gênica , Inativação Gênica , Homeostase , Camundongos , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
14.
Mov Disord ; 30(14): 1885-92, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26260437

RESUMO

BACKGROUND: This study reports the baseline characteristics of diffusion tensor imaging data in Parkinson's disease (PD) patients and healthy control subjects from the Parkinson's Progression Markers Initiative. The main goals were to replicate previous findings of abnormal diffusion imaging values from the substantia nigra. in a large multicenter cohort and determine whether nigral diffusion alterations are associated with dopamine deficits. METHODS: Two hundred twenty subjects (PD = 153; control = 67) from 10 imaging sites were included. All subjects had a full neurological exam, a ((123) I)ioflupane dopamine transporter (DAT) single-photon emission computer tomography scan, and diffusion tensor imaging. Fractional anisotropy as well as radial and axial diffusivity was computed within multiple regions across the substantia nigra. RESULTS: A repeated-measures analysis of variance found a marginally nonsignificant interaction between regional fractional anisotropy of the substantia nigra and disease status (P = 0.08), conflicting with an earlier study. However, a linear mixed model that included control regions in addition to the nigral regions revealed a significant interaction between regions and disease status (P = 0.002), implying a characteristic distribution of reduced fractional anisotropy across the substantia nigra in PD. Reduced fractional anisotropy in PD was also associated with diminished DAT binding ratios. Both axial and radial diffusivity were also abnormal in PD. CONCLUSIONS: Although routine nigral measurements of fractional anisotropy are clinically not helpful, the findings in this study suggest that more-sophisticated diffusion imaging protocols should be used when exploring the clinical utility of this imaging modality.


Assuntos
Dopamina/metabolismo , Doença de Parkinson/fisiopatologia , Substância Negra/fisiopatologia , Idoso , Imagem de Tensor de Difusão , Progressão da Doença , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Pessoa de Meia-Idade , Exame Neurológico , Doença de Parkinson/metabolismo , Substância Negra/metabolismo
15.
J Neurosci ; 35(26): 9741-53, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-26134656

RESUMO

Persistent firing of entorhinal cortex (EC) pyramidal neurons is a key component of working and spatial memory. We report here that a pro-brain-derived neurotrophic factor (proBDNF)-dependent p75NTR signaling pathway plays a major role in excitability and persistent activity of pyramidal neurons in layer V of the EC. Using electrophysiological recordings, we show that proBDNF suppresses persistent firing in entorhinal slices from wild-type mice but not from p75NTR-null mice. Conversely, function-blocking proBDNF antibodies enhance excitability of pyramidal neurons and facilitate their persistent firing, and acute exposure to function-blocking p75NTR antibodies results in enhanced firing activity of pyramidal neurons. Genetic deletion of p75NTR specifically in neurons or during adulthood also induces enhanced excitability and persistent activity, indicating that the proBDNF-p75NTR signaling cascade functions within adult neurons to inhibit pyramidal activity. Phosphatidylinositol 4,5-bisphosphate (PIP2)-sensitive transient receptor potential canonical channels play a critical role in mediating persistent firing in the EC and we hypothesized that proBDNF-dependent p75NTR activation regulates PIP2 levels. Accordingly, proBDNF decreases cholinergic calcium responses in cortical neurons and affects carbachol-induced depletion of PIP2. Further, we show that the modulation of persistent firing by proBDNF relies on a p75NTR-Rac1-PI4K pathway. The hypothesis that proBDNF and p75NTR maintain network homeostasis in the adult CNS was tested in vivo and we report that p75NTR-null mice show improvements in working memory but also display an increased propensity for severe seizures. We propose that the proBDNF-p75NTR axis controls pyramidal neuron excitability and persistent activity to balance EC performance with the risk of runaway activity. SIGNIFICANCE STATEMENT: Persistent firing of entorhinal cortex (EC) pyramidal neurons is required for working memory. We report here that pro-brain-derived neurotrophic factor (proBDNF) activates p75NTR to induce a Rac1-dependent and phosphatidylinositol 4,5-bisphosphate-dependent signaling cascade that suppresses persistent activity. Conversely, using loss-of-function approaches, we find that endogenous proBDNF or p75NTR activation strongly decreases pyramidal neuron excitability and persistent firing, suggesting that a physiological role of this proBDNF-p75NTR cascade may be to regulate working memory in vivo. Consistent with this, mice rendered null for p75NTR during adulthood show improvements in working memory but also display an increased propensity for severe seizures. We propose that by attenuating EC network performance, the proBDNF-p75NTR signaling cascade reduces the probability of epileptogenesis.


Assuntos
Potenciais de Ação/fisiologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Córtex Cerebral/citologia , Neurônios/fisiologia , Precursores de Proteínas/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Aminoquinolinas/farmacologia , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Carbacol/farmacologia , Células Cultivadas , Agonistas Colinérgicos/farmacologia , Convulsivantes/toxicidade , Modelos Animais de Doenças , Embrião de Mamíferos , Feminino , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pentilenotetrazol/toxicidade , Fosfolipase C delta/genética , Fosfolipase C delta/metabolismo , Pilocarpina/toxicidade , Precursores de Proteínas/genética , Precursores de Proteínas/farmacologia , Pirimidinas/farmacologia , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/imunologia , Convulsões/induzido quimicamente , Convulsões/genética , Convulsões/fisiopatologia
16.
Neurotoxicology ; 48: 239-48, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25929683

RESUMO

BACKGROUND: We previously found evidence of reduced gray and white matter volume in Gulf War (GW) veterans with predicted low-level exposure to sarin (GB) and cyclosarin (GF). Because loss of white matter tissue integrity has been linked to both gray and white matter atrophy, the current study sought to test the hypothesis that GW veterans with predicted GB/GF exposure have evidence of disrupted white matter microstructural integrity. METHODS: Measures of fractional anisotropy and directional (i.e., axial and radial) diffusivity were assessed from the 4T diffusion tensor images (DTI) of 59 GW veterans with predicted GB/GF exposure and 59 "matched" unexposed GW veterans (mean age: 48 ± 7 years). The DTI data were analyzed using regions of interest (ROI) analyses that accounted for age, sex, total brain gray and white matter volume, trauma exposure, posttraumatic stress disorder, current major depression, and chronic multisymptom illness status. RESULTS: There were no significant group differences in fractional anisotropy or radial diffusivity. However, there was increased axial diffusivity in GW veterans with predicted GB/GF exposure compared to matched, unexposed veterans throughout the brain, including the temporal stem, corona radiata, superior and inferior (hippocampal) cingulum, inferior and superior fronto-occipital fasciculus, internal and external capsule, and superficial cortical white matter blades. Post hoc analysis revealed significant correlations between higher fractional anisotropy and lower radial diffusivity with better neurobehavioral performance in unexposed GW veterans. In contrast, only increased axial diffusivity in posterior limb of the internal capsule was associated with better psychomotor function in GW veterans with predicted GB/GF exposure. CONCLUSIONS: The finding that increased axial diffusivity in a region of the brain that contains descending corticospinal fibers was associated with better psychomotor function and the lack of significant neurobehavioral deficits in veterans with predicted GB/GF exposure hint at the possibility that the widespread increases in axial diffusivity that we observed in GW veterans with predicted GB/GF exposure relative to unexposed controls may reflect white matter reorganization after brain injury (i.e., exposure to GB/GF).


Assuntos
Substâncias para a Guerra Química/efeitos adversos , Guerra do Golfo , Leucoencefalopatias/induzido quimicamente , Compostos Organofosforados/efeitos adversos , Sarina/efeitos adversos , Saúde dos Veteranos , Substância Branca/efeitos dos fármacos , Adulto , Anisotropia , Estudos de Casos e Controles , Imagem de Tensor de Difusão , Feminino , Humanos , Exposição por Inalação/efeitos adversos , Leucoencefalopatias/patologia , Leucoencefalopatias/fisiopatologia , Leucoencefalopatias/psicologia , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Desempenho Psicomotor/efeitos dos fármacos , Substância Branca/patologia , Substância Branca/fisiopatologia
17.
Mov Disord ; 30(9): 1229-36, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25920732

RESUMO

BACKGROUND: Parkinson's disease (PD) is histopathologically characterized by the loss of dopamine neurons in the substantia nigra pars compacta. The depletion of these neurons is thought to reduce the dopaminergic function of the nigrostriatal pathway, as well as the neural fibers that link the substantia nigra to the striatum (putamen and caudate), causing a dysregulation in striatal activity that ultimately leads to lack of movement control. Based on diffusion tensor imaging, visualizing this pathway and measuring alterations of the fiber integrity remain challenging. The objectives were to 1) develop a diffusion tensor tractography protocol for reliably tracking the nigrostriatal fibers on multicenter data; 2) test whether the integrities measured by diffusion tensor imaging of the nigrostriatal fibers are abnormal in PD; and 3) test whether abnormal integrities of the nigrostriatal fibers in PD patients are associated with the severity of motor disability and putaminal dopamine binding ratios. METHODS: Diffusion tensor tractography was performed on 50 drug-naïve PD patients and 27 healthy control subjects from the international multicenter Parkinson's Progression Marker Initiative. RESULTS: Tractography consistently detected the nigrostriatal fibers, yielding reliable diffusion measures. Fractional anisotropy, along with radial and axial diffusivity of the nigrostriatal tract, showed systematic abnormalities in patients. In addition, variations in fractional anisotropy and radial diffusivity of the nigrostriatal tract were associated with the degree of motor deficits in PD patients. CONCLUSION: Taken together, the findings imply that the diffusion tensor imaging characteristic of the nigrostriatal tract is potentially an index for detecting and staging of early PD.


Assuntos
Corpo Estriado/patologia , Imagem de Tensor de Difusão , Vias Neurais/fisiopatologia , Doença de Parkinson/patologia , Parte Compacta da Substância Negra/patologia , Idoso , Anisotropia , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Pessoa de Meia-Idade , Vias Neurais/patologia , Reprodutibilidade dos Testes , Índice de Gravidade de Doença
18.
J Neurosci ; 35(7): 3240-7, 2015 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-25698758

RESUMO

The medial temporal lobe is implicated as a key brain region involved in the pathogenesis of Alzheimer's disease (AD) and consequent memory loss. Tau tangle aggregation in this region may develop concurrently with cortical Aß deposition in preclinical AD, but the pathological relationship between tau and Aß remains unclear. We used task-free fMRI with a focus on the medical temporal lobe, together with Aß PET imaging, in cognitively normal elderly human participants. We found that cortical Aß load was related to disrupted intrinsic functional connectivity of the perirhinal cortex, which is typically the first brain region affected by tauopathies in AD. There was no concurrent association of cortical Aß load with cognitive performance or brain atrophy. These findings suggest that dysfunction in the medial temporal lobe may represent a very early sign of preclinical AD and may predict future memory loss.


Assuntos
Envelhecimento/patologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Lobo Temporal/irrigação sanguínea , Lobo Temporal/diagnóstico por imagem , Idoso , Mapeamento Encefálico , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Oxigênio/sangue , Tomografia por Emissão de Pósitrons
19.
Neurotoxicology ; 44: 263-9, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25058901

RESUMO

BACKGROUND: More than 100,000 US troops were potentially exposed to chemical warfare agents sarin (GB) and cyclosarin (GF) when an ammunition dump at Khamisiyah, Iraq was destroyed during the 1991 Gulf War (GW). We previously reported reduced hippocampal volume in GW veterans with suspected GB/GF exposure relative to matched, unexposed GW veterans estimated from 1.5T magnetic resonance images (MRI). Here we investigate, in a different cohort of GW veterans, whether low-level GB/GF exposure is associated with structural alterations in specific hippocampal subfields, estimated from 4T MRI. METHODS: The Automatic Segmentation of Hippocampal Subfields (ASHS) technique was used to quantify CA1, CA2, CA3 and dentate gyrus (DG), and subiculum (SUB) subfields volumes from high-resolution T2-weighted images acquired on a 4T MR scanner in 56 GW veterans with suspected GB/GF exposure and 56 "matched" unexposed GW veterans (mean age 49±7 years). RESULTS: GB/GF exposed veterans had smaller CA2 (p=0.003) and CA3/DG (p=0.01) subfield volumes compared to matched, unexposed GW veterans. There were no group difference in total hippocampal volume, quantified with FreeSurfer, and no dose-response relationship between estimated levels of GB/GF exposure and total hippocampal or subfield volume. CONCLUSIONS: These findings extend our previous report of structural alterations in the hippocampi of GW veterans with suspected GB/GF exposure to volume changes in the CA2, CA3, and DG hippocampal subfields in a different cohort of GW veterans with suspected GB/GF exposure.


Assuntos
Substâncias para a Guerra Química/toxicidade , Hipocampo/efeitos dos fármacos , Compostos Organofosforados/toxicidade , Sarina/toxicidade , Feminino , Guerra do Golfo , Hipocampo/patologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Veteranos
20.
Alzheimers Dement ; 10(3 Suppl): S146-54, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24924666

RESUMO

Posttraumatic stress disorder (PTSD) is associated with smaller volumes of the hippocampus, as has been demonstrated by meta-analyses. Proposed mechanistic relationships are reviewed briefly, including the hypothesis that sleep disturbances mediate the effects of PTSD on hippocampal volume. Evidence for this includes findings that insomnia and restricted sleep are associated with changes in hippocampal cell regulation and impairments in cognition. We present results of a new study of 187 subjects in whom neither PTSD nor poor sleep was associated with lower hippocampal volume. We outline a broad research agenda centered on the hypothesis that sleep changes mediate the relationship between PTSD and hippocampal volume.


Assuntos
Hipocampo/patologia , Distúrbios do Início e da Manutenção do Sono/complicações , Distúrbios do Início e da Manutenção do Sono/patologia , Transtornos de Estresse Pós-Traumáticos/complicações , Transtornos de Estresse Pós-Traumáticos/patologia , Feminino , Guerra do Golfo , Humanos , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Autorrelato , Índice de Gravidade de Doença , Sono , Inquéritos e Questionários , Veteranos
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