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1.
iScience ; 24(11): 103256, 2021 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-34761180

RESUMO

Neutrophil extracellular traps (NETs) have been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS) driven by viruses or bacteria, as well as in numerous immune-mediated disorders. Histone citrullination by the enzyme peptidylarginine deiminase 4 (PAD4) and the consequent decondensation of chromatin are hallmarks in the induction of NETs. Nevertheless, additional histone modifications that may govern NETosis are largely overlooked. Herein, we show that histone deacetylases (HDACs) play critical roles in driving NET formation in human and mouse neutrophils. HDACs belonging to the zinc-dependent lysine deacetylases family are necessary to deacetylate histone H3, thus allowing the activity of PAD4 and NETosis. Of note, HDAC inhibition in mice protects against microbial-induced pneumonia and septic shock, decreasing NETosis and inflammation. Collectively, our findings illustrate a new fundamental step that governs the release of NETs and points to HDAC inhibitors as therapeutic agents that may be used to protect against ARDS and sepsis.

2.
Mol Neurobiol ; 2021 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-34757590

RESUMO

Understanding the mechanisms underlying amyotrophic lateral sclerosis (ALS) is crucial for the development of new therapies. Previous studies have demonstrated that mitochondrial dysfunction is a key pathogenetic event in ALS. Interestingly, studies in Alzheimer's disease (AD) post-mortem brain and animal models link alterations in mitochondrial function to interactions between hyperphosphorylated tau and dynamin-related protein 1 (DRP1), the GTPase involved in mitochondrial fission. Recent evidence suggest that tau may be involved in ALS pathogenesis, therefore, we sought to determine whether hyperphosphorylated tau may lead to mitochondrial fragmentation and dysfunction in ALS and whether reducing tau may provide a novel therapeutic approach. Our findings demonstrated that pTau-S396 is mis-localized to synapses in post-mortem motor cortex (mCTX) across ALS subtypes. Additionally, the treatment with ALS synaptoneurosomes (SNs), enriched in pTau-S396, increased oxidative stress, induced mitochondrial fragmentation, and altered mitochondrial connectivity without affecting cell survival in vitro. Furthermore, pTau-S396 interacted with DRP1, and similar to pTau-S396, DRP1 accumulated in SNs across ALS subtypes, suggesting increases in mitochondrial fragmentation in ALS. As previously reported, electron microscopy revealed a significant decrease in mitochondria density and length in ALS mCTX. Lastly, reducing tau levels with QC-01-175, a selective tau degrader, prevented ALS SNs-induced mitochondrial fragmentation and oxidative stress in vitro. Collectively, our findings suggest that increases in pTau-S396 may lead to mitochondrial fragmentation and oxidative stress in ALS and decreasing tau may provide a novel strategy to mitigate mitochondrial dysfunction in ALS. pTau-S396 mis-localizes to synapses in ALS. ALS synaptoneurosomes (SNs), enriched in pTau-S396, increase oxidative stress and induce mitochondrial fragmentation in vitro. pTau-S396 interacts with the pro-fission GTPase DRP1 in ALS. Reducing tau with a selective degrader, QC-01-175, mitigates ALS SNs-induced mitochondrial fragmentation and increases in oxidative stress in vitro.

3.
J Med Chem ; 64(19): 14745-14756, 2021 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-34549949

RESUMO

To better understand the role of bromodomain and extra-terminal domain (BET) proteins in epigenetic mechanisms, we developed a series of thienodiazepine-based derivatives and identified two compounds, 3a and 6a, as potent BET inhibitors. Further in vivo pharmacokinetic studies and analysis of in vitro metabolic stability of 6a revealed excellent brain penetration and reasonable metabolic stability. Compounds 3a and 6a were radiolabeled with fluorine-18 in two steps and utilized in positron emission tomography (PET) imaging studies in mice. Preliminary PET imaging results demonstrated that [18F]3a and [18F]6a have good brain uptake (with maximum SUV = 1.7 and 2, respectively) and binding specificity in mice brains. These results show that [18F]6a is a potential PET radiotracer that could be applied to imaging BET proteins in the brain. Further optimization and improvement of the metabolic stability of [18F]6a are still needed in order to create optimal PET imaging probes of BET family members.

4.
Sci Rep ; 11(1): 17029, 2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34426604

RESUMO

Mutations in MAPT (microtubule-associated protein tau) cause frontotemporal dementia (FTD). MAPT mutations are associated with abnormal tau phosphorylation levels and accumulation of misfolded tau protein that can propagate between neurons ultimately leading to cell death (tauopathy). Recently, a p.A152T tau variant was identified as a risk factor for FTD, Alzheimer's disease, and synucleinopathies. Here we used induced pluripotent stem cells (iPSC) from a patient carrying this p.A152T variant to create a robust, functional cellular assay system for probing pathophysiological tau accumulation and phosphorylation. Using stably transduced iPSC-derived neural progenitor cells engineered to enable inducible expression of the pro-neural transcription factor Neurogenin 2 (Ngn2), we generated disease-relevant, cortical-like glutamatergic neurons in a scalable, high-throughput screening compatible format. Utilizing automated confocal microscopy, and an advanced image-processing pipeline optimized for analysis of morphologically complex human neuronal cultures, we report quantitative, subcellular localization-specific effects of multiple kinase inhibitors on tau, including ones under clinical investigation not previously reported to affect tau phosphorylation. These results demonstrate the potential for using patient iPSC-derived ex vivo models of tauopathy as genetically accurate, disease-relevant systems to probe tau biochemistry and support the discovery of novel therapeutics for tauopathies.


Assuntos
Glutamatos/metabolismo , Processamento de Imagem Assistida por Computador , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Neurônios/patologia , Proteômica , Tauopatias/patologia , Proteínas tau/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores/metabolismo , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Piridinas/química , Piridinas/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
5.
Cell ; 184(17): 4547-4563.e17, 2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34314701

RESUMO

Frontotemporal dementia (FTD) because of MAPT mutation causes pathological accumulation of tau and glutamatergic cortical neuronal death by unknown mechanisms. We used human induced pluripotent stem cell (iPSC)-derived cerebral organoids expressing tau-V337M and isogenic corrected controls to discover early alterations because of the mutation that precede neurodegeneration. At 2 months, mutant organoids show upregulated expression of MAPT, glutamatergic signaling pathways, and regulators, including the RNA-binding protein ELAVL4, and increased stress granules. Over the following 4 months, mutant organoids accumulate splicing changes, disruption of autophagy function, and build-up of tau and P-tau-S396. By 6 months, tau-V337M organoids show specific loss of glutamatergic neurons as seen in individuals with FTD. Mutant neurons are susceptible to glutamate toxicity, which can be rescued pharmacologically by the PIKFYVE kinase inhibitor apilimod. Our results demonstrate a sequence of events that precede neurodegeneration, revealing molecular pathways associated with glutamate signaling as potential targets for therapeutic intervention in FTD.

6.
PLoS One ; 16(7): e0252048, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264955

RESUMO

Neurofibromatosis Type 2 (NF2) is an autosomal dominant genetic syndrome caused by mutations in the NF2 tumor suppressor gene resulting in multiple schwannomas and meningiomas. There are no FDA approved therapies for these tumors and their relentless progression results in high rates of morbidity and mortality. Through a combination of high throughput screens, preclinical in vivo modeling, and evaluation of the kinome en masse, we identified actionable drug targets and efficacious experimental therapeutics for the treatment of NF2 related schwannomas and meningiomas. These efforts identified brigatinib (ALUNBRIG®), an FDA-approved inhibitor of multiple tyrosine kinases including ALK, to be a potent inhibitor of tumor growth in established NF2 deficient xenograft meningiomas and a genetically engineered murine model of spontaneous NF2 schwannomas. Surprisingly, neither meningioma nor schwannoma cells express ALK. Instead, we demonstrate that brigatinib inhibited multiple tyrosine kinases, including EphA2, Fer and focal adhesion kinase 1 (FAK1). These data demonstrate the power of the de novo unbiased approach for drug discovery and represents a major step forward in the advancement of therapeutics for the treatment of NF2 related malignancies.


Assuntos
Neoplasias Meníngeas/genética , Meningioma/genética , Neurilemoma/genética , Neurofibromina 2/deficiência , Neurofibromina 2/genética , Compostos Organofosforados/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Proliferação de Células , Humanos , Mutação , Neurilemoma/patologia
7.
J Neurochem ; 158(5): 1058-1073, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34077555

RESUMO

Activity-regulated cytoskeleton-associated protein (Arc) is an immediate early gene product that support neuroplastic changes important for cognitive function and memory formation. As a protein with homology to the retroviral Gag protein, a particular characteristic of Arc is its capacity to self-assemble into virus-like capsids that can package mRNAs and transfer those transcripts to other cells. Although a lot has been uncovered about the contributions of Arc to neuron biology and behavior, very little is known about how different functions of Arc are coordinately regulated both temporally and spatially in neurons. The answer to this question we hypothesized must involve the occurrence of different protein post-translational modifications acting to confer specificity. In this study, we used mass spectrometry and sequence prediction strategies to map novel Arc phosphorylation sites. Our approach led us to recognize serine 67 (S67) and threonine 278 (T278) as residues that can be modified by TNIK, which is a kinase abundantly expressed in neurons that shares many functional overlaps with Arc and has, along with its interacting proteins such as the NMDA receptor, and been implicated as a risk factor for psychiatric disorders. Furthermore, characterization of each residue using site-directed mutagenesis to create S67 and T278 mutant variants revealed that TNIK action at those amino acids can strongly influence Arc's subcellular distribution and self-assembly as capsids. Together, our findings reveal an unsuspected connection between Arc and TNIK. Better understanding of the interplay between these two proteins in neuronal cells could lead to new insights about apparition and progression of psychiatric disorders. Cover Image for this issue: https://doi.org/10.1111/jnc.15077.


Assuntos
Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Camundongos , Neurônios/metabolismo , Fosforilação/fisiologia
8.
ACS Chem Neurosci ; 12(2): 271-284, 2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33417763

RESUMO

Genomic instability caused by a deficiency in the DNA damage response and repair has been linked to age-related cognitive decline and neurodegenerative diseases. Preventing genomic instability that ultimately leads to neuronal death may provide a broadly effective strategy to protect against multiple potential genotoxic stressors. Recently, the zinc-dependent class I histone deacetylase (HDAC1) has been identified as a critical factor for protecting neurons from deleterious effects of DNA damage in Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). Translating these observations to a novel neuroprotective therapy for AD, ALS, and FTD may be advanced by the identification of small molecules capable of increasing the deacetylase activity of HDAC1 selectively over other structurally similar HDACs. Here, we demonstrate that exifone, a drug previously shown to be effective in treating cognitive deficits associated with AD and Parkinson's disease, the molecular mechanism of which has remained poorly understood, potently activates the deacetylase activity of HDAC1. We show that exifone acts as a mixed, nonessential activator of HDAC1 that is capable of binding to both free and substrate-bound enzyme, resulting in an increased relative maximal rate of HDAC1-catalyzed deacetylation. Exifone can directly bind to HDAC1 based upon biolayer interferometry assays with kinetic and selectivity profiling, suggesting that HDAC1 is preferentially targeted compared to other class I HDACs and the kinase CDK5, which have also been implicated in neurodegeneration. Consistent with a mechanism of deacetylase activation intracellularly, the treatment of human induced pluripotent stem cell (iPSC)-derived neuronal cells resulted in globally decreased histone acetylation. Moreover, exifone treatment was neuroprotective in a tauopathy patient iPSC-derived neuronal model subject to oxidative stress. Taken together, these findings reveal exifone as a potent activator of HDAC1-mediated deacetylation, thereby offering a lead for novel therapeutic development aiming to protect genomic integrity in the context of neurodegeneration and aging.


Assuntos
Histona Desacetilases , Células-Tronco Pluripotentes Induzidas , Benzofenonas , Histona Desacetilase 1 , Humanos , Neurônios
9.
Mol Psychiatry ; 26(1): 168-185, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32636474

RESUMO

Given its chronicity, contribution to disability and morbidity, and prevalence of more than 2%, the effective treatment, and prevention of bipolar disorder represents an area of significant unmet medical need. While more than half a century has passed since the introduction of lithium into widespread use at the birth of modern psychopharmacology, that medication remains a mainstay for the acute treatment and prevention of recurrent mania/hypomania and depression that characterize bipolar disorder. However, the continued limited understanding of how lithium modulates affective behavior and lack of validated cellular and animal models have resulted in obstacles to discovering more effective mood stabilizers with fewer adverse side effects. In particular, while there has been progress in developing new pharmacotherapy for mania, developing effective treatments for acute bipolar depression remain inadequate. Recent large-scale human genetic studies have confirmed the complex, polygenic nature of the risk architecture of bipolar disorder, and its overlap with other major neuropsychiatric disorders. Such discoveries have begun to shed light on the pathophysiology of bipolar disorder. Coupled with broader advances in human neurobiology, neuropharmacology, noninvasive neuromodulation, and clinical trial design, we can envision novel therapeutic strategies informed by defined molecular mechanisms and neural circuits and targeted to the root cause of the pathophysiology. Here, we review recent advances toward the goal of better treatments for bipolar disorder, and we outline major challenges for the field of translational neuroscience that necessitate continued focus on fundamental research and discovery.


Assuntos
Transtorno Bipolar/tratamento farmacológico , Transtorno Bipolar/genética , Medicina de Precisão/tendências , Anticonvulsivantes/uso terapêutico , Antimaníacos/uso terapêutico , Humanos , Compostos de Lítio/uso terapêutico
10.
Int J Mol Sci ; 21(23)2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33255694

RESUMO

Tauopathies are neurodegenerative diseases characterized by the pathological accumulation of microtubule-associated protein tau (MAPT) in the form of neurofibrillary tangles and paired helical filaments in neurons and glia, leading to brain cell death. These diseases include frontotemporal dementia (FTD) and Alzheimer's disease (AD) and can be sporadic or inherited when caused by mutations in the MAPT gene. Despite an incredibly high socio-economic burden worldwide, there are still no effective disease-modifying therapies, and few tau-focused experimental drugs have reached clinical trials. One major hindrance for therapeutic development is the knowledge gap in molecular mechanisms of tau-mediated neuronal toxicity and death. For the promise of precision medicine for brain disorders to be fulfilled, it is necessary to integrate known genetic causes of disease, i.e., MAPT mutations, with an understanding of the dysregulated molecular pathways that constitute potential therapeutic targets. Here, the growing understanding of known and proposed mechanisms of disease etiology will be reviewed, together with promising experimental tau-directed therapeutics, such as recently developed tau degraders. Current challenges faced by the fields of tau research and drug discovery will also be addressed.


Assuntos
Neuroglia/metabolismo , Neurônios/metabolismo , Tauopatias/genética , Proteínas tau/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Morte Celular/genética , Citoesqueleto/genética , Citoesqueleto/patologia , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Humanos , Mutação/genética , Emaranhados Neurofibrilares/genética , Emaranhados Neurofibrilares/patologia , Neuroglia/patologia , Neurônios/patologia , Tauopatias/patologia
11.
ACS Chem Neurosci ; 11(24): 4128-4138, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33253521

RESUMO

Glycogen synthase kinase 3-beta (GSK3ß) is a critical regulator of several cellular pathways involved in neurodevelopment and neuroplasticity and as such is a potential focus for the discovery of new neurotherapeutics toward the treatment of neuropsychiatric and neurodegenerative diseases. The majority of efforts to develop inhibitors of GSK3ß have been focused on developing small molecule inhibitors that compete with adenosine triphosphate (ATP) through direct interaction with the ATP binding site. This strategy has presented selectivity challenges due to the evolutionary conservation of this domain within the kinome. The disrupted in schizophrenia 1 (DISC1) protein has previously been shown to bind and inhibit GSK3ß activity. Here, we report the characterization of a 44-mer peptide derived from human DISC1 (hDISCtide) that is sufficient to both bind and inhibit GSK3ß in a noncompetitive mode distinct from classical ATP competitive inhibitors. Based on multiple independent biochemical and biophysical assays, we propose that hDISCtide interacts at two distinct regions of GSK3ß: an inhibitory region that partially overlaps with the binding site of FRATide, a well-known GSK3ß binding peptide, and a specific binding region that is unique to hDISCtide. Taken together, our findings present a novel avenue for developing a peptide-based selective inhibitor of GSK3ß.


Assuntos
Glicogênio Sintase Quinase 3 beta , Proteínas do Tecido Nervoso , Humanos , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/farmacologia , Fosforilação
12.
Alzheimers Dement (N Y) ; 6(1): e12090, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33083513

RESUMO

As knowledge of Alzheimer's disease (AD) progression improves, the field has recognized the need to diversify the pipeline, broaden strategies and approaches to therapies, as well as delivery mechanisms. A better understanding of the earliest biological processes of AD/dementia would help inform drug target selection. Currently there are a number of programs exploring these alternate avenues. This meeting will allow experts in the field (academia, industry, government) to provide perspectives and experiences that can help elucidate what the pipeline looks like today and what avenues hold promise in developing new therapies across the stages of AD. The focus here is on Active Immunotherapies and Alternative Therapeutic Modalities. This topic includes active vaccines, antisense oligomers, and cell-based therapy among others, and highlights new clinical developments that utilize these modalities.

13.
Elife ; 92020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32990597

RESUMO

Somatic expansion of the Huntington's disease (HD) CAG repeat drives the rate of a pathogenic process ultimately resulting in neuronal cell death. Although mechanisms of toxicity are poorly delineated, transcriptional dysregulation is a likely contributor. To identify modifiers that act at the level of CAG expansion and/or downstream pathogenic processes, we tested the impact of genetic knockout, in HttQ111 mice, of Hdac2 or Hdac3 in medium-spiny striatal neurons that exhibit extensive CAG expansion and exquisite disease vulnerability. Both knockouts moderately attenuated CAG expansion, with Hdac2 knockout decreasing nuclear huntingtin pathology. Hdac2 knockout resulted in a substantial transcriptional response that included modification of transcriptional dysregulation elicited by the HttQ111 allele, likely via mechanisms unrelated to instability suppression. Our results identify novel modifiers of different aspects of HD pathogenesis in medium-spiny neurons and highlight a complex relationship between the expanded Htt allele and Hdac2 with implications for targeting transcriptional dysregulation in HD.


Assuntos
Corpo Estriado/fisiopatologia , Histona Desacetilase 2/genética , Histona Desacetilases/genética , Doença de Huntington/genética , Neurônios/fisiologia , Animais , Núcleo Celular , Modelos Animais de Doenças , Histona Desacetilase 2/metabolismo , Histona Desacetilases/metabolismo , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/enzimologia , Camundongos , Camundongos Endogâmicos C57BL
14.
Nat Commun ; 11(1): 3258, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591533

RESUMO

Tauopathies are neurodegenerative diseases associated with accumulation of abnormal tau protein in the brain. Patient iPSC-derived neuronal cell models replicate disease-relevant phenotypes ex vivo that can be pharmacologically targeted for drug discovery. Here, we explored autophagy as a mechanism to reduce tau burden in human neurons and, from a small-molecule screen, identify the mTOR inhibitors OSI-027, AZD2014 and AZD8055. These compounds are more potent than rapamycin, and robustly downregulate phosphorylated and insoluble tau, consequently reducing tau-mediated neuronal stress vulnerability. MTORC1 inhibition and autophagy activity are directly linked to tau clearance. Notably, single-dose treatment followed by washout leads to a prolonged reduction of tau levels and toxicity for 12 days, which is mirrored by a sustained effect on mTORC1 inhibition and autophagy. This new insight into the pharmacodynamics of mTOR inhibitors in regulation of neuronal autophagy may contribute to development of therapies for tauopathies.


Assuntos
Autofagia , Neurônios/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Estresse Fisiológico , Tauopatias/metabolismo , Proteínas tau/metabolismo , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Pessoa de Meia-Idade , Modelos Biológicos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurônios/efeitos dos fármacos , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Fenótipo , Ratos Wistar , Estresse Fisiológico/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Tauopatias/patologia , Fatores de Tempo
15.
Nat Commun ; 11(1): 2484, 2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32424276

RESUMO

DNA damage contributes to brain aging and neurodegenerative diseases. However, the factors stimulating DNA repair to stave off functional decline remain obscure. We show that HDAC1 modulates OGG1-initated 8-oxoguanine (8-oxoG) repair in the brain. HDAC1-deficient mice display age-associated DNA damage accumulation and cognitive impairment. HDAC1 stimulates OGG1, a DNA glycosylase known to remove 8-oxoG lesions that are associated with transcriptional repression. HDAC1 deficiency causes impaired OGG1 activity, 8-oxoG accumulation at the promoters of genes critical for brain function, and transcriptional repression. Moreover, we observe elevated 8-oxoG along with reduced HDAC1 activity and downregulation of a similar gene set in the 5XFAD mouse model of Alzheimer's disease. Notably, pharmacological activation of HDAC1 alleviates the deleterious effects of 8-oxoG in aged wild-type and 5XFAD mice. Our work uncovers important roles for HDAC1 in 8-oxoG repair and highlights the therapeutic potential of HDAC1 activation to counter functional decline in brain aging and neurodegeneration.


Assuntos
Envelhecimento/patologia , Doença de Alzheimer/patologia , Encéfalo/patologia , Dano ao DNA , DNA Glicosilases/metabolismo , Histona Desacetilase 1/metabolismo , Estresse Oxidativo , Acetilação , Envelhecimento/genética , Doença de Alzheimer/complicações , Doença de Alzheimer/fisiopatologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Sequência de Bases , Benzofenonas/farmacologia , Cognição/efeitos dos fármacos , Transtornos Cognitivos/complicações , Transtornos Cognitivos/patologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Ontologia Genética , Guanina/análogos & derivados , Guanina/metabolismo , Memória/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Regiões Promotoras Genéticas/genética
16.
Nucl Med Biol ; 84-85: 96-101, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32320910

RESUMO

INTRODUCTION: Bromodomain and extra-terminal domain (BET) family proteins play a vital role in the epigenetic regulation process by interacting with acetylated lysine (Ac-K) residues in histones. BET inhibitors have become promising candidates to treat various diseases through the inhibition of the interaction between BET bromodomains and Ac-K of histone tails. With a molecular imaging probe, noninvasive imaging such as positron emission tomography (PET) can visualize the distribution and roles of BET family proteins in vivo and enlighten our understanding of BET protein function in both healthy and diseased tissue. METHODS: We radiolabeled the potent BET inhibitor INCB054329 by N-methylation to make [11C]PB003 as a BET PET radiotracer. The bioactivity evaluation of unlabeled PB003 in vitro was performed to confirm its binding affinity for BRDs, then the PET/CT imaging in rodents was performed to evaluate the bioactivity of [11C]PB003 in vivo. RESULTS: In our in vitro evaluation, PB003 showed a high BET binding affinity for BRDs (Kd = 2 nM, 1.2 nM, and 1.2 nM for BRD2, BRD3, and BRD4, respectively). In vivo PET/CT imaging demonstrated that [11C]PB003 has favorable uptake with appropriate kinetics and distributions in main peripheral organs. Besides, the blockade of [11C]PB003 binding was found in our blocking study which indicated the specificity of [11C]PB003. However, the BBB penetration and brain uptake of [11C]PB003 was limited, with only a maximum 0.2% injected dose/g at ~2 min post-injection. CONCLUSION: The imaging results in rodents in vivo demonstrate that [11C]PB003 binds to BET with high selectivity and specificity and has favorable uptake in peripheral organs. However, the low brain uptake of [11C]PB003 limits the visualization of brain regions indicating the efforts are still needed to discover the new BET imaging probes for brain visualization.


Assuntos
Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Marcação por Isótopo , Cinética , Metilação , Domínios Proteicos , Traçadores Radioativos , Radioquímica
17.
Transl Psychiatry ; 10(1): 76, 2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-32094324

RESUMO

The effective treatment of bipolar disorder (BD) represents a significant unmet medical need. Although lithium remains a mainstay of treatment for BD, limited knowledge regarding how it modulates affective behavior has proven an obstacle to discovering more effective mood stabilizers with fewer adverse side effects. One potential mechanism of action of lithium is through inhibition of the serine/threonine protein kinase GSK3ß, however, relevant substrates whose change in phosphorylation may mediate downstream changes in neuroplasticity remain poorly understood. Here, we used human induced pluripotent stem cell (hiPSC)-derived neuronal cells and stable isotope labeling by amino acids in cell culture (SILAC) along with quantitative mass spectrometry to identify global changes in the phosphoproteome upon inhibition of GSK3α/ß with the highly selective, ATP-competitive inhibitor CHIR-99021. Comparison of phosphorylation changes to those induced by therapeutically relevant doses of lithium treatment led to the identification of collapsin response mediator protein 2 (CRMP2) as being highly sensitive to both treatments as well as an extended panel of structurally distinct GSK3α/ß inhibitors. On this basis, a high-content image-based assay in hiPSC-derived neurons was developed to screen diverse compounds, including FDA-approved drugs, for their ability to mimic lithium's suppression of CRMP2 phosphorylation without directly inhibiting GSK3ß kinase activity. Systemic administration of a subset of these CRMP2-phosphorylation suppressors were found to mimic lithium's attenuation of amphetamine-induced hyperlocomotion in mice. Taken together, these studies not only provide insights into the neural substrates regulated by lithium, but also provide novel human neuronal assays for supporting the development of mechanism-based therapeutics for BD and related neuropsychiatric disorders.


Assuntos
Transtorno Bipolar , Células-Tronco Pluripotentes Induzidas , Anfetamina/farmacologia , Animais , Transtorno Bipolar/tratamento farmacológico , Humanos , Lítio/farmacologia , Compostos de Lítio/farmacologia , Camundongos , Fosforilação
18.
Mol Autism ; 11(1): 2, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31921404

RESUMO

Background: Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with frequent occurrence of epilepsy, autism spectrum disorder (ASD), intellectual disability (ID), and tumors in multiple organs. The aberrant activation of mTORC1 in TSC has led to treatment with mTORC1 inhibitor rapamycin as a lifelong therapy for tumors, but TSC-associated neurocognitive manifestations remain unaffected by rapamycin. Methods: Here, we generated patient-specific, induced pluripotent stem cells (iPSCs) from a TSC patient with a heterozygous, germline, nonsense mutation in exon 15 of TSC1 and established an isogenic set of heterozygous (Het), null and corrected wildtype (Corr-WT) iPSCs using CRISPR/Cas9-mediated gene editing. We differentiated these iPSCs into neural progenitor cells (NPCs) and examined neurodevelopmental phenotypes, signaling and changes in gene expression by RNA-seq. Results: Differentiated NPCs revealed enlarged cell size in TSC1-Het and Null NPCs, consistent with mTORC1 activation. TSC1-Het and Null NPCs also revealed enhanced proliferation and altered neurite outgrowth in a genotype-dependent manner, which was not reversed by rapamycin. Transcriptome analyses of TSC1-NPCs revealed differentially expressed genes that display a genotype-dependent linear response, i.e., genes upregulated/downregulated in Het were further increased/decreased in Null. In particular, genes linked to ASD, epilepsy, and ID were significantly upregulated or downregulated warranting further investigation. In TSC1-Het and Null NPCs, we also observed basal activation of ERK1/2, which was further activated upon rapamycin treatment. Rapamycin also increased MNK1/2-eIF4E signaling in TSC1-deficient NPCs. Conclusion: MEK-ERK and MNK-eIF4E pathways regulate protein translation, and our results suggest that aberrant translation distinct in TSC1/2-deficient NPCs could play a role in neurodevelopmental defects. Our data showing upregulation of these signaling pathways by rapamycin support a strategy to combine a MEK or a MNK inhibitor with rapamycin that may be superior for TSC-associated CNS defects. Importantly, our generation of isogenic sets of NPCs from TSC patients provides a valuable platform for translatome and large-scale drug screening studies. Overall, our studies further support the notion that early developmental events such as NPC proliferation and initial process formation, such as neurite number and length that occur prior to neuronal differentiation, represent primary events in neurogenesis critical to disease pathogenesis of neurodevelopmental disorders such as ASD.


Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células-Tronco Neurais/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Esclerose Tuberosa , Sistemas CRISPR-Cas , Códon sem Sentido , Edição de Genes , Mutação em Linhagem Germinativa , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Neurogênese , Fenótipo , RNA-Seq , Transdução de Sinais , Sirolimo , Proteína 1 do Complexo Esclerose Tuberosa/genética
19.
Mol Psychiatry ; 25(11): 2712-2727, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-31988434

RESUMO

Although circular RNAs (circRNAs) are enriched in the mammalian brain, very little is known about their potential involvement in brain function and psychiatric disease. Here, we show that circHomer1a, a neuronal-enriched circRNA abundantly expressed in the frontal cortex, derived from Homer protein homolog 1 (HOMER1), is significantly reduced in both the prefrontal cortex (PFC) and induced pluripotent stem cell-derived neuronal cultures from patients with schizophrenia (SCZ) and bipolar disorder (BD). Moreover, alterations in circHomer1a were positively associated with the age of onset of SCZ in both the dorsolateral prefrontal cortex (DLPFC) and orbitofrontal cortex (OFC). No correlations between the age of onset of SCZ and linear HOMER1 mRNA were observed, whose expression was mostly unaltered in BD and SCZ postmortem brain. Using in vivo circRNA-specific knockdown of circHomer1a in mouse PFC, we show that it modulates the expression of numerous alternative mRNA transcripts from genes involved in synaptic plasticity and psychiatric disease. Intriguingly, in vivo circHomer1a knockdown in mouse OFC resulted in specific deficits in OFC-mediated cognitive flexibility. Lastly, we demonstrate that the neuronal RNA-binding protein HuD binds to circHomer1a and can influence its synaptic expression in the frontal cortex. Collectively, our data uncover a novel psychiatric disease-associated circRNA that regulates synaptic gene expression and cognitive flexibility.


Assuntos
Transtorno Bipolar/genética , Cognição , Regulação da Expressão Gênica , RNA Circular/genética , Esquizofrenia/genética , Sinapses/metabolismo , Adulto , Animais , Feminino , Proteínas de Arcabouço Homer/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Córtex Pré-Frontal/metabolismo
20.
Ann N Y Acad Sci ; 1471(1): 18-56, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-30875083

RESUMO

Development of effective therapeutics for neurological disorders has historically been challenging partly because of lack of accurate model systems in which to investigate disease etiology and test new therapeutics at the preclinical stage. Human stem cells, particularly patient-derived induced pluripotent stem cells (iPSCs) upon differentiation, have the ability to recapitulate aspects of disease pathophysiology and are increasingly recognized as robust scalable systems for drug discovery. We review advances in deriving cellular models of human central nervous system (CNS) disorders using iPSCs along with strategies for investigating disease-relevant phenotypes, translatable biomarkers, and therapeutic targets. Given their potential to identify novel therapeutic targets and leads, we focus on phenotype-based, small-molecule screens employing human stem cell-derived models. Integrated efforts to assemble patient iPSC-derived cell models with deeply annotated clinicopathological data, along with molecular and drug-response signatures, may aid in the stratification of patients, diagnostics, and clinical trial success, shifting translational science and precision medicine approaches. A number of remaining challenges, including the optimization of cost-effective, large-scale culture of iPSC-derived cell types, incorporation of aging into neuronal models, as well as robustness and automation of phenotypic assays to support quantitative drug efficacy, toxicity, and metabolism testing workflows, are covered. Continued advancement of the field is expected to help fully humanize the process of CNS drug discovery.


Assuntos
Doenças do Sistema Nervoso Central/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Medicina de Precisão , Diferenciação Celular/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/patologia , Doenças do Sistema Nervoso Central/genética , Doenças do Sistema Nervoso Central/patologia , Descoberta de Drogas , Humanos , Neurônios/efeitos dos fármacos
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