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1.
FASEB J ; 38(6): e23556, 2024 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-38498348

RESUMO

PARP-1 over-activation results in cell death via excessive PAR generation in different cell types, including neurons following brain ischemia. Glycolysis, mitochondrial function, and redox balance are key cellular processes altered in brain ischemia. Studies show that PAR generated after PARP-1 over-activation can bind hexokinase-1 (HK-1) and result in glycolytic defects and subsequent mitochondrial dysfunction. HK-1 is the neuronal hexokinase and catalyzes the first reaction of glycolysis, converting glucose to glucose-6-phosphate (G6P), a common substrate for glycolysis, and the pentose phosphate pathway (PPP). PPP is critical in maintaining NADPH and GSH levels via G6P dehydrogenase activity. Therefore, defects in HK-1 will not only decrease cellular bioenergetics but will also cause redox imbalance due to the depletion of GSH. In brain ischemia, whether PAR-mediated inhibition of HK-1 results in bioenergetics defects and redox imbalance is not known. We used oxygen-glucose deprivation (OGD) in mouse cortical neurons to mimic brain ischemia in neuronal cultures and observed that PARP-1 activation via PAR formation alters glycolysis, mitochondrial function, and redox homeostasis in neurons. We used pharmacological inhibition of PARP-1 and adenoviral-mediated overexpression of wild-type HK-1 (wtHK-1) and PAR-binding mutant HK-1 (pbmHK-1). Our data show that PAR inhibition or overexpression of HK-1 significantly improves glycolysis, mitochondrial function, redox homeostasis, and cell survival in mouse cortical neurons exposed to OGD. These results suggest that PAR binding and inhibition of HK-1 during OGD drive bioenergetic defects in neurons due to inhibition of glycolysis and impairment of mitochondrial function.


Assuntos
Isquemia Encefálica , Oxigênio , Camundongos , Animais , Oxigênio/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/metabolismo , Glucose/metabolismo , Isquemia Encefálica/metabolismo , Glicólise , Neurônios/metabolismo , Oxirredução
2.
Stroke ; 55(4): 983-989, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38482715

RESUMO

BACKGROUND: There is limited research on outcomes of patients with posttraumatic stress disorder (PTSD) who also develop stroke, particularly regarding racial disparities. Our goal was to determine whether PTSD is associated with the risk of hospital readmission after stroke and whether racial disparities existed. METHODS: The analytical sample consisted of all veterans receiving care in the Veterans Health Administration who were identified as having a new stroke requiring inpatient admission based on the International Classification of Diseases codes. PTSD and comorbidities were identified using the International Classification of Diseases codes and given the date of first occurrence. The retrospective cohort data were obtained from the Veterans Affairs Corporate Data Warehouse. The main outcome was any readmission to Veterans Health Administration with a stroke diagnosis. The hypothesis that PTSD is associated with readmission after stroke was tested using Cox regression adjusted for patient characteristics including age, sex, race, PTSD, smoking status, alcohol use, and comorbidities treated as time-varying covariates. RESULTS: Our final cohort consisted of 93 651 patients with inpatient stroke diagnosis and no prior Veterans Health Administration codes for stroke starting from 1999 with follow-up through August 6, 2022. Of these patients, 12 916 (13.8%) had comorbid PTSD. Of the final cohort, 16 896 patients (18.0%) with stroke were readmitted. Our fully adjusted model for readmission found an interaction between African American veterans and PTSD with a hazard ratio of 1.09 ([95% CI, 1.00-1.20] P=0.047). In stratified models, PTSD has a significant hazard ratio of 1.10 ([95% CI, 1.02-1.18] P=0.01) for African American but not White veterans (1.05 [95% CI, 0.99-1.11]; P=0.10). CONCLUSIONS: Among African American veterans who experienced stroke, preexisting PTSD was associated with increased risk of readmission, which was not significant among White veterans. This study highlights the need to focus on high-risk groups to reduce readmissions after stroke.


Assuntos
Transtornos de Estresse Pós-Traumáticos , Acidente Vascular Cerebral , Veteranos , Humanos , Estados Unidos/epidemiologia , Transtornos de Estresse Pós-Traumáticos/epidemiologia , Transtornos de Estresse Pós-Traumáticos/diagnóstico , Estudos Retrospectivos , Readmissão do Paciente , Acidente Vascular Cerebral/epidemiologia , Acidente Vascular Cerebral/terapia , Comorbidade
3.
Glia ; 71(3): 485-508, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36380708

RESUMO

A major hallmark of neuroinflammation is the activation of microglia and astrocytes with the induction of inflammatory mediators such as IL-1ß, TNF-α, iNOS, and IL-6. Neuroinflammation contributes to disease progression in a plethora of neurological disorders ranging from acute CNS trauma to chronic neurodegenerative disease. Posttranscriptional pathways of mRNA stability and translational efficiency are major drivers for the expression of these inflammatory mediators. A common element in this level of regulation centers around the adenine- and uridine-rich element (ARE) which is present in the 3' untranslated region (UTR) of the mRNAs encoding these inflammatory mediators. (ARE)-binding proteins (AUBPs) such as Human antigen R (HuR), Tristetraprolin (TTP) and KH- type splicing regulatory protein (KSRP) are key nodes for directing these posttranscriptional pathways and either promote (HuR) or suppress (TTP and KSRP) glial production of inflammatory mediators. This review will discuss basic concepts of ARE-mediated RNA regulation and its impact on glial-driven neuroinflammatory diseases. We will discuss strategies to target this novel level of gene regulation for therapeutic effect and review exciting preliminary studies that underscore its potential for treating neurological disorders.


Assuntos
Doenças do Sistema Nervoso Central , Doenças Neurodegenerativas , Humanos , RNA/metabolismo , Doenças Neuroinflamatórias , Doenças Neurodegenerativas/metabolismo , Astrócitos/metabolismo , Doenças do Sistema Nervoso Central/genética , Doenças do Sistema Nervoso Central/terapia , Doenças do Sistema Nervoso Central/metabolismo , Mediadores da Inflamação/metabolismo
4.
J Neurochem ; 164(5): 643-657, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36527420

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that selectively attacks motor neurons, and leads to progressive muscle weakness and death. A common pathological feature is the misfolding, aggregation, and cytoplasmic mislocalization of TAR DNA-binding protein 43 (TDP-43) proteins in more than 95% of ALS patients, suggesting a universal role TDP-43 proteinopathy in ALS. Mutations in SQSTM1/p62 have been identified in familial and sporadic cases of ALS. MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate their target genes. Emerging evidence indicates that miRNA dysregulation is associated with neuronal toxicity and mitochondrial dysfunction, and also plays a pivotal role in ALS pathogenesis. Here, we report the first evidence that miR-183-5p is aberrantly upregulated in spinal cords of patients with ALS. Using luciferase reporter assays and miR-183-5p agomirs, we demonstrate that miR-183-5p regulates the SQSTM1/p62 3'-untranslated region to suppress expression. A miR-183-5p agomir attenuated SOSTM1/p62 expression and led to an increase in TDP-43 protein levels in neuronal and non-neuronal cells. In contrast, a miR-183-5p antagomir decreased TDP-43 but increased SQSTM1/p62 protein levels. The antagomir repressed formation of stress granules and aggregated TDP43 protein in neuronal cells under stress-induced conditions and protected against cytotoxicity. Knockdown of SQSTM1/p62 decreased total ubiquitination and increased TDP-43 protein aggregation, indicating that SQSTM1/p62 may play a protective role in cells. In summary, our study reveals a novel mechanism of TDP-43 proteinopathy mediated by the miR-183-5p and provides a molecular link between aberrant RNA processing and protein degradation, two major pillars in ALS pathogenesis.


Assuntos
Esclerose Lateral Amiotrófica , MicroRNAs , Doenças Neurodegenerativas , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Proteína Sequestossoma-1/metabolismo , Doenças Neurodegenerativas/metabolismo , Antagomirs/metabolismo , Neurônios Motores/metabolismo , MicroRNAs/metabolismo , Proteínas de Ligação a DNA/metabolismo
5.
Glia ; 70(1): 155-172, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34533864

RESUMO

Glial activation with the production of pro-inflammatory mediators is a major driver of disease progression in neurological processes ranging from acute traumatic injury to chronic neurodegenerative diseases such as amyotrophic lateral sclerosis and Alzheimer's disease. Posttranscriptional regulation is a major gateway for glial activation as many mRNAs encoding pro-inflammatory mediators contain adenine- and uridine-rich elements (ARE) in the 3' untranslated region which govern their expression. We have previously shown that HuR, an RNA regulator that binds to AREs, plays a major positive role in regulating inflammatory cytokine production in glia. HuR is predominantly nuclear in localization but translocates to the cytoplasm to exert a positive regulatory effect on RNA stability and translational efficiency. Homodimerization of HuR is necessary for translocation and we have developed a small molecule inhibitor, SRI-42127, that blocks this process. Here we show that SRI-42127 suppressed HuR translocation in LPS-activated glia in vitro and in vivo and significantly attenuated the production of pro-inflammatory mediators including IL1ß, IL-6, TNF-α, iNOS, CXCL1, and CCL2. Cytokines typically associated with anti-inflammatory effects including TGF-ß1, IL-10, YM1, and Arg1 were either unaffected or minimally affected. SRI-42127 suppressed microglial activation in vivo and attenuated the recruitment/chemotaxis of neutrophils and monocytes. RNA kinetic studies and luciferase studies indicated that SRI-42127 has inhibitory effects both on mRNA stability and gene promoter activation. In summary, our findings underscore HuR's critical role in promoting glial activation and the potential for SRI-42127 and other HuR inhibitors for treating neurological diseases driven by this activation.


Assuntos
Proteína Semelhante a ELAV 1 , Lipopolissacarídeos , Regiões 3' não Traduzidas , Proteínas ELAV/genética , Proteínas ELAV/metabolismo , Proteína Semelhante a ELAV 1/genética , Humanos , Cinética , Lipopolissacarídeos/toxicidade , Doenças Neuroinflamatórias
6.
Int J Mol Sci ; 23(14)2022 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-35886863

RESUMO

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease characterized by skeletal muscle instability, progressive muscle wasting, and fibrosis. A major driver of DMD pathology stems from aberrant upregulation of transforming growth factor ß (TGFß) signaling. In this report, we investigated the major transducers of TGFß signaling, i.e., receptor Smads (R-Smads), in DMD patient skeletal muscle and observed a 48-fold increase in Smad8 mRNA. Smad1, Smad2, Smad3, and Smad5 mRNA were only minimally increased. A similar pattern was observed in the muscle from the mdx5cv mouse. Western blot analysis showed upregulation of phosphorylated Smad1, Smad5, and Smad8 compared to total Smad indicating activation of this pathway. In parallel, we observed a profound diminishment of muscle-enriched microRNAs (myomiRs): miR-1, miR-133a, and miR-133b. The pattern of Smad8 induction and myomiR suppression was recapitulated in C2C12 muscle cells after stimulation with bone morphogenetic protein 4 (BMP4), a signaling factor that we found upregulated in DMD muscle. Silencing Smad8 in C2C12 myoblasts derepressed myomiRs and promoted myoblast differentiation; there was also a concomitant upregulation of myogenic regulatory factors (myogenin and myocyte enhancer factor 2D) and suppression of a pro-inflammatory cytokine (interleukin-6). Our data suggest that Smad8 is a negative regulator of miR-1, miR-133a, and miR-133b in muscle cells and that the BMP4-Smad8 axis is a driver of dystrophic pathology in DMD.


Assuntos
MicroRNAs , Distrofia Muscular de Duchenne , Proteína Smad8 , Animais , Camundongos , Camundongos Endogâmicos mdx , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , RNA Mensageiro/metabolismo , Proteína Smad8/genética , Proteína Smad8/metabolismo , Fator de Crescimento Transformador beta/metabolismo
7.
Glia ; 68(6): 1165-1181, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31859421

RESUMO

Distal axonopathy is a recognized pathological feature of amyotrophic lateral sclerosis (ALS). In the peripheral nerves of ALS patients, motor axon loss elicits a Wallerian-like degeneration characterized by denervated Schwann cells (SCs) together with immune cell infiltration. However, the pathogenic significance of denervated SCs accumulating following impaired axonal growth in ALS remains unclear. Here, we analyze SC phenotypes in sciatic nerves of ALS patients and paralytic SOD1G93A rats, and identify remarkably similar and specific reactive SC phenotypes based on the pattern of S100ß, GFAP, isolectin and/or p75NTR immunoreactivity. Different subsets of reactive SCs expressed colony-stimulating factor-1 (CSF1) and Interleukin-34 (IL-34) and closely interacted with numerous endoneurial CSF-1R-expressing monocyte/macrophages, suggesting a paracrine mechanism of myeloid cell expansion and activation. SCs bearing phagocytic phenotypes as well as endoneurial macrophages expressed stem cell factor (SCF), a trophic factor that attracts and activates mast cells through the c-Kit receptor. Notably, a subpopulation of Ki67+ SCs expressed c-Kit in the sciatic nerves of SOD1G93A rats, suggesting a signaling pathway that fuels SC proliferation in ALS. c-Kit+ mast cells were also abundant in the sciatic nerve from ALS donors but not in controls. Pharmacological inhibition of CSF-1R and c-Kit with masitinib in SOD1G93A rats potently reduced SC reactivity and immune cell infiltration in the sciatic nerve and ventral roots, suggesting a mechanism by which the drug ameliorates peripheral nerve pathology. These findings provide strong evidence for a previously unknown inflammatory mechanism triggered by SCs in ALS peripheral nerves that has broad application in developing novel therapies.


Assuntos
Esclerose Lateral Amiotrófica/patologia , Inflamação/metabolismo , Interleucinas/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Células de Schwann/metabolismo , Fator de Células-Tronco/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Animais , Axônios/patologia , Modelos Animais de Doenças , Humanos , Masculino , Neurônios Motores/patologia , Neuroglia/metabolismo , Ratos Transgênicos
8.
Glia ; 67(12): 2424-2439, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31400163

RESUMO

Glioblastoma is a malignant brain tumor that portends a poor prognosis. Its resilience, in part, is related to a remarkable capacity for manipulating the microenvironment to promote its growth and survival. Microglia/macrophages are prime targets, being drawn into the tumor and stimulated to produce factors that support tumor growth and evasion from the immune system. Here we show that the RNA regulator, HuR, plays a key role in the tumor-promoting response of microglia/macrophages. Knockout (KO) of HuR led to reduced tumor growth and proliferation associated with prolonged survival in a murine model of glioblastoma. Analysis of tumor composition by flow cytometry showed that tumor-associated macrophages (TAMs) were decreased, more polarized toward an M1-like phenotype, and had reduced PD-L1 expression. There was an overall increase in infiltrating CD4+ cells, including Th1 and cytotoxic effector cells, and a concomitant reduction in tumor-associated polymorphonuclear myeloid-derived suppressor cells. Molecular and cellular analyses of HuR KO TAMs and cultured microglia showed changes in migration, chemoattraction, and chemokine/cytokine profiles that provide potential mechanisms for the altered tumor microenvironment and reduced tumor growth in HuR KO mice. In summary, HuR is a key modulator of pro-glioma responses by microglia/macrophages through the molecular regulation of chemokines, cytokines, and other factors. Our findings underscore the relevance of HuR as a therapeutic target in glioblastoma.


Assuntos
Neoplasias Encefálicas/imunologia , Proteína Semelhante a ELAV 1/deficiência , Deleção de Genes , Glioma/imunologia , Macrófagos/imunologia , Microglia/imunologia , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Proteína Semelhante a ELAV 1/genética , Glioma/genética , Glioma/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microglia/metabolismo , Microglia/patologia , Microambiente Tumoral/fisiologia
9.
Int J Mol Sci ; 20(16)2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31395804

RESUMO

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of upper and lower motor neurons accompanied by proliferation of reactive microglia in affected regions. However, it is unknown whether the hematopoietic marker CD34 can identify a subpopulation of proliferating microglial cells in the ALS degenerating spinal cord. Immunohistochemistry for CD34 and microglia markers was performed in lumbar spinal cords of ALS rats bearing the SOD1G93A mutation and autopsied ALS and control human subjects. Characterization of CD34-positive cells was also performed in primary cell cultures of the rat spinal cords. CD34 was expressed in a large number of cells that closely interacted with degenerating lumbar spinal cord motor neurons in symptomatic SOD1G93A rats, but not in controls. Most CD34+ cells co-expressed the myeloid marker CD11b, while only a subpopulation was stained for Iba1 or CD68. Notably, CD34+ cells actively proliferated and formed clusters adjacent to damaged motor neurons bearing misfolded SOD1. CD34+ cells were identified in the proximity of motor neurons in autopsied spinal cord from sporadic ALS subjects but not in controls. Cell culture of symptomatic SOD1G93A rat spinal cords yielded a large number of CD34+ cells exclusively in the non-adherent phase, which generated microglia after successive passaging. A yet unrecognized CD34+ cells, expressing or not the microglial marker Iba1, proliferate and accumulate adjacent to degenerating spinal motor neurons, representing an intriguing cell target for approaching ALS pathogenesis and therapeutics.


Assuntos
Esclerose Lateral Amiotrófica/patologia , Antígenos CD34/análise , Microglia/patologia , Neurônios Motores/patologia , Esclerose Lateral Amiotrófica/genética , Animais , Proliferação de Células , Células Cultivadas , Humanos , Masculino , Microglia/citologia , Mutação Puntual , Dobramento de Proteína , Ratos , Medula Espinal/patologia , Superóxido Dismutase-1/análise , Superóxido Dismutase-1/genética
10.
Neurobiol Dis ; 114: 85-94, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29486297

RESUMO

ALS is a fatal neurodegenerative disorder of motor neurons leading to progressive atrophy and weakness of muscles. Some of the earliest pathophysiological changes occur at the level of skeletal muscle and the neuromuscular junction. We previously identified distinct mRNA patterns, including members of the Smad and TGF-ß family, that emerge in muscle tissue at the earliest (pre-clinical) stages. These patterns track disease progression in the mutant SOD1 mouse and are present in human ALS muscle. Because miRNAs play a direct regulatory role in mRNA expression, we hypothesized in this study that there would be distinct miRNA patterns in ALS muscle appearing in early stages that could track disease progression. We performed next-generation miRNA sequencing on muscle samples from G93A SOD1 mice at early (pre-clinical) and late (symptomatic) stages, and identified distinct miRNA patterns at both stages with some overlap. An Ingenuity Pathway Analysis predicted effects on a number of pathways relevant to ALS including TGF-ß signaling, axon guidance signaling, and mitochondrial function. A subset of miRNAs was validated in the G93A SOD1 mouse at four stages of disease, and several appeared to track disease progression, including miR-206. We assessed these miRNAs in a large cohort of human ALS and disease control samples and found that some had similar changes but were not specific for ALS. Surprisingly, miR-206 levels did not change overall compared to normal controls, but did correlate with changes in strength of the muscle biopsied. In summary, we identified distinct miRNA patterns in ALS muscle that reflected disease stage which could potentially be used as biomarkers of disease activity.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Progressão da Doença , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo , Superóxido Dismutase/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Biomarcadores/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Superóxido Dismutase/genética
11.
Glia ; 65(6): 945-963, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28300326

RESUMO

In neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), chronic activation of microglia contributes to disease progression. Activated microglia produce cytokines, chemokines, and other factors that normally serve to clear infection or damaged tissue either directly or through the recruitment of other immune cells. The molecular program driving this phenotype is classically linked to the transcription factor NF-κB and characterized by the upregulation of proinflammatory factors such as IL-1ß, TNF-α, and IL-6. Here, we investigated the role of HuR, an RNA-binding protein that regulates gene expression through posttranscriptional pathways, on the molecular and cellular phenotypes of activated microglia. We performed RNA sequencing of HuR-silenced microglia and found significant attenuation of lipopolysaccharide-induced IL-1ß and TNF-α inflammatory pathways and other factors that promote microglial migration and invasion. RNA kinetics and luciferase reporter studies suggested that the attenuation was related to altered promoter activity rather than a change in RNA stability. HuR-silenced microglia showed reduced migration, invasion, and chemotactic properties but maintained viability. MMP-12, a target exquisitely sensitive to HuR knockdown, participates in the migration/invasion phenotype. HuR is abundantly detected in the cytoplasmic compartment of activated microglia from ALS spinal cords consistent with its increased activity. Microglia from ALS-associated mutant SOD1 mice demonstrated higher migration/invasion properties which can be blocked with HuR inhibition. These findings underscore an important role for HuR in sculpting the molecular signature and phenotype of activated microglia, and as a possible therapeutic target in ALS and other neurodegenerative diseases.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Microglia/metabolismo , Adulto , Idoso , Esclerose Lateral Amiotrófica/patologia , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Células Cultivadas , Modelos Animais de Doenças , Proteína Semelhante a ELAV 1/antagonistas & inibidores , Proteína Semelhante a ELAV 1/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Metaloproteinase 12 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/patologia , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologia
12.
J Biol Chem ; 289(46): 31792-31804, 2014 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-25239623

RESUMO

Posttranscriptional gene regulation is governed by a network of RNA-binding proteins (RBPs) that interact with regulatory elements in the mRNA to modulate multiple molecular processes, including splicing, RNA transport, RNA stability, and translation. Mounting evidence indicates that there is a hierarchy within this network whereby certain RBPs cross-regulate other RBPs to coordinate gene expression. HuR, an RNA-binding protein we linked previously to aberrant VEGF mRNA metabolism in models of SOD1-associated amyotrophic lateral sclerosis, has been identified as being high up in this hierarchy, serving as a regulator of RNA regulators. Here we investigated the role of HuR in regulating two RBPs, TDP-43 and FUS/TLS, that have been linked genetically to amyotrophic lateral sclerosis. We found that HuR promotes the expression of both RBPs in primary astrocytes and U251 cells under normal and stressed (hypoxic) conditions. For TDP-43, we found that HuR binds to the 3' untranslated region (UTR) and regulates its expression through translational efficiency rather than RNA stability. With HuR knockdown, there was a shift of TDP-43 and FUS mRNAs away from polysomes, consistent with translational silencing. The TDP-43 splicing function was attenuated upon HuR knockdown and could be rescued by ectopic TDP-43 lacking the 3' UTR regulatory elements. Finally, conditioned medium from astrocytes in which HuR or TDP-43 was knocked down produced significant motor neuron and cortical neuron toxicity in vitro. These findings indicate that HuR regulates TDP-43 and FUS/TLS expression and that loss of HuR-mediated RNA processing in astrocytes can alter the molecular and cellular landscape to produce a toxic phenotype.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas ELAV/metabolismo , Regulação da Expressão Gênica , Proteína FUS de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas , Animais , Astrócitos/citologia , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Meios de Cultivo Condicionados/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Proteína Semelhante a ELAV 1 , Humanos , Hipóxia , Camundongos , Neurônios Motores/metabolismo , Fenótipo , RNA/química
13.
J Neurooncol ; 118(1): 61-72, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24664369

RESUMO

Protein arginine methyltransferase 5 (PRMT5) catalyzes the formation of ω-NG,N'G-symmetric dimethylarginine residues on histones as well as other proteins. These modifications play an important role in cell differentiation and tumor cell growth. However, the role of PRMT5 in human glioma cells has not been characterized. In this study, we assessed protein expression profiles of PRMT5 in control brain, WHO grade II astrocytomas, anaplastic astrocytomas, and glioblastoma multiforme (GBM) by immunohistochemistry. PRMT5 was low in glial cells in control brain tissues and low grade astrocytomas. Its expression increased in parallel with malignant progression, and was highly expressed in GBM. Knockdown of PRMT5 by small hairpin RNA caused alterations of p-ERK1/2 and significantly repressed the clonogenic potential and viability of glioma cells. These findings indicate that PRMT5 is a marker of malignant progression in glioma tumors and plays a pivotal role in tumor growth.


Assuntos
Neoplasias Encefálicas/metabolismo , Proliferação de Células/fisiologia , Glioma/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Adulto , Idoso , Arginina/análogos & derivados , Arginina/metabolismo , Neoplasias Encefálicas/patologia , Diferenciação Celular/genética , Proliferação de Células/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Ensaio de Unidades Formadoras de Colônias , Epitélio/metabolismo , Epitélio/patologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Glioma/patologia , Humanos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Pessoa de Meia-Idade , Neurônios/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteína-Arginina N-Metiltransferases/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
14.
Neural Regen Res ; 19(4): 747-753, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-37843208

RESUMO

Amyotrophic lateral sclerosis is a fatal multisystemic neurodegenerative disease with motor neurons being a primary target. Although progressive weakness is a hallmark feature of amyotrophic lateral sclerosis, there is considerable heterogeneity, including clinical presentation, progression, and the underlying triggers for disease initiation. Based on longitudinal studies with families harboring amyotrophic lateral sclerosis-associated gene mutations, it has become apparent that overt disease is preceded by a prodromal phase, possibly in years, where compensatory mechanisms delay symptom onset. Since 85-90% of amyotrophic lateral sclerosis is sporadic, there is a strong need for identifying biomarkers that can detect this prodromal phase as motor neurons have limited capacity for regeneration. Current Food and Drug Administration-approved therapies work by slowing the degenerative process and are most effective early in the disease. Skeletal muscle, including the neuromuscular junction, manifests abnormalities at the earliest stages of the disease, before motor neuron loss, making it a promising source for identifying biomarkers of the prodromal phase. The accessibility of muscle through biopsy provides a lens into the distal motor system at earlier stages and in real time. The advent of "omics" technology has led to the identification of numerous dysregulated molecules in amyotrophic lateral sclerosis muscle, ranging from coding and non-coding RNAs to proteins and metabolites. This technology has opened the door for identifying biomarkers of disease activity and providing insight into disease mechanisms. A major challenge is correlating the myriad of dysregulated molecules with clinical or histological progression and understanding their relevance to presymptomatic phases of disease. There are two major goals of this review. The first is to summarize some of the biomarkers identified in human amyotrophic lateral sclerosis muscle that have a clinicopathological correlation with disease activity, evidence of a similar dysregulation in the SOD1G93A mouse during presymptomatic stages, and evidence of progressive change during disease progression. The second goal is to review the molecular pathways these biomarkers reflect and their potential role in mitigating or promoting disease progression, and as such, their potential as therapeutic targets in amyotrophic lateral sclerosis.

15.
bioRxiv ; 2024 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-38712206

RESUMO

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disease due to loss-of-function mutations in the DYSTROPHIN gene. DMD-related skeletal muscle wasting is typified by an aberrant immune response involving upregulation of TGFß family of cytokines. We previously demonstrated that bone morphogenetic protein 4 (BMP4) is increased in DMD and BMP4 stimulation induces a 20-fold upregulation of Smad8 transcription. However, the role of BMP4 in severely affected DMD skeletal muscle is unknown. We hypothesized that transcriptomic signatures in severely affected human DMD skeletal muscle are driven by BMP4 signaling. Transcriptomes from skeletal muscle biopsies of late-stage DMD vs. non-DMD controls and C2C12 muscle cells with or without BMP4 stimulation were generated by RNA-Seq and analyzed for single transcript differential expression as well as by Ingenuity Pathway Analysis and weighted gene co-expression network analyses. A total of 2,328 and 5,291 transcripts in the human muscle and C2C12 muscle cells, respectively, were differentially expressed. We identified an overlapping molecular signature of 1,027 genes dysregulated in DMD muscle that were induced in BMP4-stimulated C2C12 muscle cells. Highly upregulated DMD transcripts that overlapped with BMP4-stimulated C2C12 muscle cells included ADAMTS3, HCAR2, SERPING1, SMAD8 , and UNC13C. The DMD transcriptome was characterized by dysregulation of pathways involving immune function, extracellular matrix remodeling, and metabolic/mitochondrial function. In summary, we define a late-stage DMD skeletal muscle transcriptome that substantially overlaps with the BMP4-induced molecular signature in C2C12 muscle cells. This supports BMP4 as a disease-driving regulator of transcriptomic changes in late-stage DMD skeletal muscle and expands our understanding of the evolution of dystrophic signaling pathways and their associated gene networks that could be explored for therapeutic development.

16.
Brain Behav Immun Health ; 39: 100798, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39022628

RESUMO

In addition to extracellular amyloid plaques, intracellular neurofibrillary tau tangles, and inflammation, cognitive and emotional affect perturbations are characteristic of Alzheimer's disease (AD). The cognitive and emotional domains impaired by AD include several forms of decision making (such as intertemporal choice), blunted motivation (increased apathy), and impaired executive function (such as working memory and cognitive flexibility). However, the interaction between these domains of the mind and their supporting neurobiological substrates at prodromal stages of AD, or whether these interactions can be predictive of AD severity (individual variability), remain unclear. In this study, we employed a battery of cognitive and emotional tests in the young adult (5-7 mo) transgenic Fisher-344 AD (TgF344-AD; TgAD) rat model of AD. We also assessed whether markers of inflammation or AD-like pathology in the prelimbic cortex (PrL) of the medial prefrontal cortex (mPFC), basolateral amygdala (BLA), or nucleus accumbens (NAc), all structures that directly support the aforementioned behaviors, were predictive of behavioral deficits. We found TgAD rats displayed maladaptive decision making, greater apathy, and impaired working memory that was indeed predicted by AD-like pathology in the relevant brain structures, even at an early age. Moreover, we report that the BLA is an early epicenter of inflammation, and notably, AD-like pathology in the PrL, BLA, and NAc was predictive of BLA inflammation. These results suggest that operant-based battery testing may be sensitive enough to determine pathology trajectories, including neuroinflammation, from early stages of AD.

17.
bioRxiv ; 2024 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-39314333

RESUMO

Amyotrophic lateral sclerosis (ALS) is an age-related and fatal neurodegenerative disease characterized by progressive muscle weakness. There is marked heterogeneity in clinical presentation, progression, and pathophysiology with only modest treatments to slow disease progression. Molecular markers that provide insight into this heterogeneity are crucial for clinical management and identification of new therapeutic targets. In a prior muscle miRNA sequencing investigation, we identified altered FGF pathways in ALS muscle, leading us to investigate FGF21. We analyzed human ALS muscle biopsy samples and found a large increase in FGF21 expression with localization to atrophic myofibers and surrounding endomysium. A concomitant increase in FGF21 was detected in ALS spinal cords which correlated with muscle levels. FGF21 was increased in the SOD1G93A mouse beginning in presymptomatic stages. In parallel, there was dysregulation of the co-receptor, ß-Klotho. Plasma FGF21 levels were increased and high levels correlated with slower disease progression, prolonged survival, and increased body mass index. In NSC-34 motor neurons and C2C12 muscle cells expressing SOD1G93A or exposed to oxidative stress, ectopic FGF21 mitigated loss of cell viability. In summary, FGF21 is a novel biomarker in ALS that correlates with slower disease progression and exerts trophic effects under conditions of cellular stress.

18.
Muscle Nerve ; 47(1): 17-22, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23042608

RESUMO

METHODS: We compared the problems or complications associated with electrodiagnostic testing in 77 patients with implanted cardiac devices. Thirty tests were performed after magnet placement, and 47 were performed without magnet application. RESULTS: All electrodiagnostic tests were performed safely in all patients without any serious effect on the implanted cardiac devices with or without magnet placement. A significantly higher number of patient symptoms and procedure changes were reported in the magnet group (P < 0.013). No statistical difference was found in the testing difficulty or ECG changes. CONCLUSION: The magnet group patients had an approximately 11-fold greater risk of symptoms than those in the control group. Our data do not support a recommendation that magnet placement is necessary for routine electrodiagnostic testing in patients with implanted cardiac devices, as long as our general and specific guidelines are followed.


Assuntos
Desfibriladores Implantáveis/efeitos adversos , Eletromiografia/efeitos adversos , Marca-Passo Artificial/efeitos adversos , Idoso , Idoso de 80 Anos ou mais , Eletromiografia/métodos , Feminino , Humanos , Imãs , Masculino , Pessoa de Meia-Idade , Segurança do Paciente
19.
J Neurooncol ; 113(2): 195-205, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23525947

RESUMO

Malignant gliomas rely on the production of certain critical growth factors including VEGF, interleukin (IL)-6 and IL-8, to fuel rapid tumor growth, angiogenesis, and treatment resistance. Post-transcriptional regulation through adenine and uridine-rich elements of the 3' untranslated region is one mechanism for upregulating these and other growth factors. In glioma cells, we have shown that the post-transcriptional machinery is optimized for growth factor upregulation secondary to overexpression of the mRNA stabilizer, HuR. The negative regulator, tristetraprolin (TTP), on the other hand, may be suppressed because of extensive phosphorylation. Here we test that possibility by analyzing the phenotypic effects of a mutated form of TTP (mt-TTP) in which 8 phosphoserine residues were converted to alanines. We observed a significantly enhanced negative effect on growth factor expression in glioma cells at the post-transcriptional and transcriptional levels. The protein became stabilized and displayed significantly increased antiproliferative effects compared to wild-type TTP. Macroautophagy was induced with both forms of TTP, but inhibition of autophagy did not affect cell viability. We conclude that glioma cells suppress TTP function through phosphorylation of critical serine residues which in turn contributes to growth factor upregulation and tumor progression.


Assuntos
Apoptose , Neoplasias Encefálicas/patologia , Glioma/patologia , Mutação/genética , Tristetraprolina/genética , Regiões 3' não Traduzidas , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Proliferação de Células , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Glioma/genética , Glioma/metabolismo , Humanos , Imunoprecipitação , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Fosforilação , Estabilidade de RNA/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tristetraprolina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
20.
Glia ; 60(11): 1773-84, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22847996

RESUMO

Chronic inflammation in the central nervous system (CNS) is a central feature of many neurodegenerative and autoimmune diseases. As an immunologically competent cell, the astrocyte plays an important role in CNS inflammation. It is capable of expressing a number of cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1ß) that promote inflammation directly and through the recruitment of immune cells. Checkpoints are therefore in place to keep tight control over cytokine production. Adenylate/uridylate-rich elements (ARE) in the 3' untranslated region of cytokine mRNAs serve as a major checkpoint by regulating mRNA stability and translational efficiency. Here, we examined the impact of KH-type splicing regulatory protein (KSRP), an RNA binding protein which destabilizes mRNAs via the ARE, on cytokine expression and paracrine phenotypes of primary astrocytes. We identified a network of inflammatory mediators, including TNF-α and IL-1ß, whose expression increased 2 to 4-fold at the RNA level in astrocytes isolated from KSRP(-/-) mice compared to littermate controls. Upon activation, KSRP(-/-) astrocytes produced TNF-α and IL-1ß at levels that exceeded control cells by 15-fold or more. Conditioned media from KSRP(-/-) astrocytes induced chemotaxis and neuronal cell death in vitro. Surprisingly, we observed a prolongation of half-life in only a subset of mRNA targets and only after selective astrocyte activation. Luciferase reporter studies indicated that KSRP regulates cytokine gene expression at both transcriptional and post-transcriptional levels. Our results outline a critical role for KSRP in regulating pro-inflammatory mediators and have implications for a wide range of CNS inflammatory and autoimmune diseases.


Assuntos
Astrócitos/metabolismo , Morte Celular/fisiologia , Movimento Celular/fisiologia , Citocinas/biossíntese , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Animais , Regulação da Expressão Gênica , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Proteínas de Ligação a RNA/genética , Transativadores/genética , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima
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