RESUMO
Basal-like breast cancer (BLBC) is the most aggressive molecular subtype of breast cancer with worse prognosis and fewer treatment options. The underlying mechanisms upon BLBC transcriptional dysregulation and its upstream transcription factors (TFs) remain unclear. Here, among the hyperactive candidate TFs of BLBC identified by bioinformatic analysis, POU4F1 is uniquely upregulated in BLBC and is associated with poor prognosis. POU4F1 is necessary for the tumor growth and malignant phenotypes of BLBC through regulating G1/S transition by direct binding at the promoter of CDK2 and CCND1. More importantly, POU4F1 maintains BLBC identity by repressing ERα expression through CDK2-mediated EZH2 phosphorylation and subsequent H3K27me3 modification in ESR1 promoter. Knocking out POU4F1 in BLBC cells reactivates functional ERα expression, rendering BLBC sensitive to tamoxifen treatment. In-depth epigenetic analysis reveals that the subtype-specific re-configuration and activation of the bivalent chromatin in the POU4F1 promoter contributes to its unique expression in BLBC, which is maintained by DNA demethylase TET1. Together, these results reveal a subtype-specific epigenetically activated TF with critical role in promoting and maintaining BLBC, suggesting that POU4F1 is a potential therapeutic target for BLBC.
Assuntos
Neoplasias da Mama , Regulação Neoplásica da Expressão Gênica , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Humanos , Feminino , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Camundongos , Animais , Fator de Transcrição Brn-3A/genética , Fator de Transcrição Brn-3A/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Modelos Animais de Doenças , Regiões Promotoras Genéticas/genéticaRESUMO
The neural crest transcription factor BRN3A is essential for the proliferation and survival of melanoma cells. It is frequently expressed in melanoma but not in normal melanocytes or benign nevi. The mechanisms underlying the aberrant expression of BRN3A are unknown. Here, we investigated the epigenetic regulation of BRN3A in melanocytes and melanoma cell lines treated with DNA methyltransferase (DNMT), histone acetyltransferase (HAT), and histone deacetylase (HDAC) inhibitors. DNMT and HAT inhibition did not significantly alter BRN3A expression levels, whereas panHDAC inhibition by trichostatin A led to increased expression. Treatment with the isoform-specific HDAC inhibitor mocetinostat, but not with PCI-34051, also increased BRN3A expression levels, suggesting that class I HDACs HDAC1, HDAC2, and HDAC3, and class IV HDAC11, were involved in the regulation of BRN3A expression. Transient silencing of HDACs 1, 2, 3, and 11 by siRNAs revealed that, specifically, HDAC2 inhibition was able to increase BRN3A expression. ChIP-Seq analysis uncovered that HDAC2 inhibition specifically increased H3K27ac levels at a distal enhancer region of the BRN3A gene. Altogether, our data suggest that HDAC2 is a key epigenetic regulator of BRN3A in melanocytes and melanoma cells. These results highlight the importance of epigenetic mechanisms in regulating melanoma oncogenes.
Assuntos
Regulação da Expressão Gênica , Histona Desacetilase 2/metabolismo , Melanócitos/metabolismo , Melanoma/etiologia , Melanoma/metabolismo , Fator de Transcrição Brn-3A/genética , Linhagem Celular , Metilação de DNA , Epigênese Genética , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Histona Desacetilase 2/genética , Inibidores de Histona Desacetilases/farmacologia , Humanos , Melanócitos/patologia , Melanoma/patologia , Fator de Transcrição Brn-3A/metabolismoRESUMO
AIMS/HYPOTHESIS: Diabetic retinopathy is characterised by retinal neurodegeneration and retinal vascular abnormalities, affecting one third of diabetic patients with disease duration of more than 10 years. Accumulated evidence suggests that serine racemase (SR) and D-serine are correlated with the pathogenesis of diabetic retinopathy and the deletion of the Srr gene reverses neurovascular pathologies in diabetic mice. Since D-serine content is balanced by SR synthesis and D-amino acid oxidase (DAAO) degradation, we examined the roles of DAAO in diabetic retinopathy and further explored relevant therapy. METHODS: Rats were used as a model of diabetes by i.p. injection of streptozotocin at the age of 2 months and blood glucose was monitored with a glucometer. Quantitative real-time PCR was used to examine Dao mRNA and western blotting to examine targeted proteins in the retinas. Bisulphite sequencing was used to examine the methylation of Dao mRNA promoter in the retinas. Intravitreal injection of DAAO-expressing adenovirus (AAV8-DAAO) was conducted one week before streptozotocin administration. Brain specific homeobox/POU domain protein 3a (Brn3a) immunofluorescence was conducted to indicate retinal ganglion cells at 3 months after virus injection. The permeability of the blood-retinal barrier was examined by Evans blue leakage from retinal capillaries. Periodic acid-Schiff staining and haematoxylin counterstaining were used to indicate retinal vasculature, which was further examined with double immunostaining at 7 months after virus injection. RESULTS: At the age of 12 months, DAAO mRNA and protein levels in retinas from diabetic animals were reduced to 66.2% and 70.4% of those from normal (control) animals, respectively. The Dao proximal promoter contained higher levels of methylation in diabetic than in normal retinas. Consistent with the observation, DNA methyltransferase 1 was increased in diabetic retinas. Injection of DAAO-expressing virus completely prevented the loss of retinal ganglion cells and the disruption of blood-retinal barrier in diabetic rats. Diabetic retinas contained retinal ganglion cells at a density of 54 ± 4/mm2, which was restored to 68 ± 9/mm2 by DAAO overexpression, similar to the levels in normal retinas. The ratio between the number of endothelial cells and pericytes in diabetic retinas was 6.06 ± 1.93/mm2, which was reduced to 3.42 ± 0.55/mm2 by DAAO overexpression; the number of acellular capillaries in diabetic retinas was 10 ± 5/mm2, which was restored to 6 ± 2/mm2 by DAAO overexpression, similar to the levels in normal retinas. Injection of the DAAO-expressing virus increased the expression of occludin and reduced gliosis, which were examined to probe the mechanism by which the disrupted blood-retinal barrier in diabetic rats was rescued and retinal neurodegeneration was prevented. CONCLUSIONS/INTERPRETATION: Altogether, overexpression of DAAO before the onset of diabetes protects against neurovascular abnormalities in retinas from diabetic rats, which suggests a novel strategy for preventing diabetic retinopathy. Graphical abstract.
Assuntos
Barreira Hematorretiniana/enzimologia , D-Aminoácido Oxidase/biossíntese , Retinopatia Diabética/prevenção & controle , Células Ganglionares da Retina/enzimologia , Animais , Barreira Hematorretiniana/patologia , Permeabilidade Capilar , D-Aminoácido Oxidase/genética , Metilação de DNA , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/enzimologia , Retinopatia Diabética/enzimologia , Retinopatia Diabética/etiologia , Retinopatia Diabética/patologia , Indução Enzimática , Masculino , Degeneração Neural , Regiões Promotoras Genéticas , Ratos Sprague-Dawley , Células Ganglionares da Retina/patologia , Fator de Transcrição Brn-3A/genética , Fator de Transcrição Brn-3A/metabolismoRESUMO
Traumatic optic neuropathy (TON) is characterized by visual dysfunction after indirect or direct injury to the optic nerve following blunt head trauma. TON is associated with increased oxidative stress and inflammation resulting in retinal ganglion cell (RGC) death. Remote ischemic post-conditioning (RIC) has been shown to enhance endogenous protective mechanisms in diverse disease models including stroke, vascular cognitive impairment (VCI), retinal injury and optic nerve injury. However, the protective mechanisms underlying the improvement of retinal function and RGC survival after RIC treatment remain unclear. Here, we hypothesized that RIC therapy may be protective following TON by preventing RGC death, oxidative insult and inflammation in the mouse retina. To carry out the study, mice were divided in three different groups (Control, TON and TON + RIC). We harvested retinal tissue 5 days after TON induction for western blotting and histochemical analysis. We observed increased TON-induced retinal cell death compared with controls by cleaved caspase-3 immunohistochemistry. Furthermore, the TON cohort demonstrated increased TUNEL positive cells which were significantly attenuated by RIC. Immunofluorescence data showed that oxidative stress markers dihydroethidium (DHE), NOX-2 and nitrotyrosine expression were elevated in the TON group relative to controls and RIC therapy significantly reduced the expression level of these markers. Next, we found that the proinflammatory cytokine TNF-α was increased and anti-inflammatory IL-10 was decreased in plasma of TON animals, and RIC therapy reversed this expression level. Interestingly, western blotting of retinal tissue showed that RGC marker Brn3a and tight junction proteins (ZO-1 and Occludin), and AMPKα1 expression were downregulated in the TON group compared to controls. However, RIC significantly increased the expression levels of these proteins. Together these data suggest that RIC therapy activates endogenous protective mechanisms which may attenuate TON-induced oxidative stress and inflammation, and improves BRB integrity.
Assuntos
Pós-Condicionamento Isquêmico , Traumatismos do Nervo Óptico/terapia , Adenilato Quinase/biossíntese , Adenilato Quinase/genética , Animais , Barreira Hematorretiniana , Caspase 3/biossíntese , Caspase 3/genética , Morte Celular , Proteínas do Olho/biossíntese , Proteínas do Olho/genética , Membro Posterior/irrigação sanguínea , Interleucina-10/sangue , Isquemia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/fisiologia , Modelos Animais , NADPH Oxidase 2/análise , Doenças Neuroinflamatórias/etiologia , Doenças Neuroinflamatórias/prevenção & controle , Estresse Oxidativo , Células Ganglionares da Retina/patologia , Superóxidos/análise , Fator de Transcrição Brn-3A/biossíntese , Fator de Transcrição Brn-3A/genética , Fator de Necrose Tumoral alfa/sangue , Tirosina/análogos & derivados , Tirosina/análiseRESUMO
Aberrant promoter methylation and ensuing abnormal gene expression are important epigenetic mechanisms that contribute to colorectal oncogenesis. Yet, the prognostic significance of such methylation-driven genes in colorectal cancer (CRC) remains obscure. Herein, a total of 181 genes were identified as the methylation-driven molecular features of CRC by integrated analysis of the expression profiles and the matched DNA methylation data from The Cancer Genome Atlas (TCGA) database. Among them, a five-gene signature (POU4F1, NOVA1, MAGEA1, SLCO4C1, and IZUMO2) was developed as a risk assessment model for predicting the clinical outcomes in CRC. The Kaplan-Meier analysis and Harrell's C index demonstrated that the risk assessment model significantly distinguished the patients in high or low-risk groups (p-value < 0.0001 log-rank test, HR: 2.034, 95% CI: 1.419-2.916, C index: 0.655). The sensitivity and specificity were validated by the receiver operating characteristic (ROC) analysis. Furthermore, different pharmaceutical treatment responses were observed between the high-risk and low-risk groups. Indeed, the methylation-driven gene signature could act as an independent prognostic evaluation biomarker for assessing the OS of CRC patients and guiding the pharmaceutical treatment. Compared with known biomarkers, the methylation-driven gene signature could reveal cross-omics molecular features for improving clinical stratification and prognosis.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Metilação de DNA , Epigênese Genética , Perfilação da Expressão Gênica , Transcriptoma , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/terapia , Bases de Dados Genéticas , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Masculino , Antígenos Específicos de Melanoma/genética , Pessoa de Meia-Idade , Antígeno Neuro-Oncológico Ventral , Transportadores de Ânions Orgânicos/genética , Valor Preditivo dos Testes , Prognóstico , Proteínas de Ligação a RNA/genética , Medição de Risco , Fatores de Risco , Fator de Transcrição Brn-3A/genéticaRESUMO
Over-expression of the human epidermal growth factor receptor-2 (HER2) is related to aggressive tumors and poor prognosis in breast cancer. Trastuzumab (TRA) resistance leads to tumor recurrence and metastasis, resulting in poor prognosis in HER2-positive breast cancer. POU Class 4 Homeobox 1 (POU4F1) is a member of the POU domain family transcription factors, and has a key role in regulating cancers. However, its effects on TRA-resistant HER2-positive breast cancer are still vague. In the present study, we found that POU4F1 expression was dramatically increased in clinical breast cancer specimens with TRA resistance. Higher POU4F1 was also detected in HER2-positive breast cancer cells with TRA resistance than that of the parental ones. Poor prognosis was detected in breast cancer patients with high POU4F1 expression. Under TRA treatment, POU4F1 knockdown significantly reduced the proliferative capacity of HER2-positive breast cancer cells with TRA resistance. POU4F1 silence also sensitized resistant HER-positive breast cancer cells to TRA treatment in vivo using a xenograft mouse model, along with the markedly reduced tumor growth rate and tumor weight. Moreover, we found that POU4F1 deletion greatly decreased the activation of mitogen-activated or extracellular signal-regulated protein kinase kinases 1 and 2 (MEK1/2) and extracellular-regulated kinase 1/2 (ERK1/2) signaling pathways in breast cancer cells with TRA resistance. Migration and invasion were also effectively hindered by POU4F1 knockdown in TRA-resistant HER2-positive breast cancer cells. Notably, we found that POU4F1 deletion-improved chemosensitivity of HER2-positive breast cancer cells with drug-resistance to TRA treatment was closely associated with the blockage of ERK1/2 signaling. Collectively, our findings reported a critical role of POU4F1 in regulating TRA resistance, and demonstrated the underlying molecular mechanisms in HER2-positive breast cancer. Thus, POU4F1 may be a promising prognostic and therapeutic target to develop effective treatment for overcoming TRA resistance.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Neoplasias da Mama/metabolismo , Sistema de Sinalização das MAP Quinases , Fator de Transcrição Brn-3A/metabolismo , Trastuzumab/uso terapêutico , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Camundongos Nus , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Invasividade Neoplásica , Receptor ErbB-2/análise , Fator de Transcrição Brn-3A/genética , Fator de Transcrição Brn-3A/fisiologiaRESUMO
Unresolved inflammation can lead to tissue fibrosis and impaired organ function. Macrophage-myofibroblast transition (MMT) is one newly identified mechanism by which ongoing chronic inflammation causes progressive fibrosis in different forms of kidney disease. However, the mechanisms underlying MMT are still largely unknown. Here, we discovered a brain-specific homeobox/POU domain protein Pou4f1 (Brn3a) as a specific regulator of MMT. Interestingly, we found that Pou4f1 is highly expressed by macrophages undergoing MMT in sites of fibrosis in human and experimental kidney disease, identified by coexpression of the myofibroblast marker, α-SMA. Unexpectedly, Pou4f1 expression peaked in the early stage in renal fibrogenesis in vivo and during MMT of bone marrow-derived macrophages (BMDMs) in vitro. Mechanistically, chromatin immunoprecipitation (ChIP) assay identified that Pou4f1 is a Smad3 target and the key downstream regulator of MMT, while microarray analysis defined a Pou4f1-dependent fibrogenic gene network for promoting TGF-ß1/Smad3-driven MMT in BMDMs at the transcriptional level. More importantly, using two mouse models of progressive renal interstitial fibrosis featuring the MMT process, we demonstrated that adoptive transfer of TGF-ß1-stimulated BMDMs restored both MMT and renal fibrosis in macrophage-depleted mice, which was prevented by silencing Pou4f1 in transferred BMDMs. These findings establish a role for Pou4f1 in MMT and renal fibrosis and suggest that Pou4f1 may be a therapeutic target for chronic kidney disease with progressive renal fibrosis.
Assuntos
Proteína Smad3/metabolismo , Fator de Transcrição Brn-3A/genética , Fator de Crescimento Transformador beta1/metabolismo , Animais , Feminino , Fibrose/fisiopatologia , Redes Reguladoras de Genes , Humanos , Inflamação/patologia , Rim/patologia , Nefropatias/genética , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/metabolismo , Transdução de Sinais/genética , Fator de Transcrição Brn-3A/metabolismo , Fator de Transcrição Brn-3A/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Sistema Urinário/metabolismoRESUMO
Traumatic optic neuropathy is a common clinical problem. Damage to the optic nerve leads to shear stress and triggers secondary swelling within the optic canal. The study aims to explore the role of the inflammatory response following optic nerve injury (ONI) in toll-like receptor-9 knockout mice (TLR-9-/-) compared to wild-type mice (WT). At first, TLR-9-/- and WT mice were subjected to ONI. We then found that ONI significantly up-regulated TLR-9 expression levels in retinal tissues of WT mice. The retinal degeneration after ONI was alleviated in TLR-9-/- mice, as evidenced by the increased number of retinal ganglion cells (RGCs) and thickness of inner retinal layer (IRL). TUNEL staining and immunofluorescence staining of BRN3A indicated that TLR-9 knockout effectively improved the survival of RGCs. ONI-enhanced expression of Iba-1 and TMEM119 was markedly reduced in TLR-9-/- mice, indicating the suppression of microglial activation. Moreover, production of pro-inflammatory regulators, including inducible nitric oxide synthase (iNOS), macrophage chemo-attractant protein (MCP)-1, cyclooxygenase-2 (COX-2), interleukin (IL)-1ß, IL-18 and tumor necrosis factor-α (TNF-α), was significantly decreased in TLR-9-/- mice following ONI. TLR-9 knockout-attenuated inflammation was mainly through repressing myeloid differentiation factor 88 (MyD88) and IL-1 receptor-associated kinase 4 (IRAK4). Furthermore, ONI greatly up-regulated the protein expression levels of phosphorylated (p)-IKKα, p-IκBα and p-nuclear factor (NF)-κB, whereas being repressed in TLR-9-/- mice. The effects of TLR-9 on ONI were verified in lipopolysaccharide (LPS)-stimulated retinal microglial cells transfected with small interfering RNA TLR-9 (siTLR-9). As expected, promoting TLR-9 with its agonist markedly restored inflammation in TLR-9 knockdown cells stimulated by LPS. Therefore, all findings above suggested that suppressing TLR-9 showed neuroprotective effects against ONI through reducing inflammatory response, and TILR-9 might be a promising therapeutic target to develop effective strategies for the treatment of optic neuropathies.
Assuntos
Microglia/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Traumatismos do Nervo Óptico/genética , Nervo Óptico/metabolismo , Células Ganglionares da Retina/metabolismo , Receptor Toll-Like 9/genética , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Contagem de Células , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Regulação da Expressão Gênica , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Inflamação , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Interleucina-18/genética , Interleucina-18/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microglia/patologia , Fator 88 de Diferenciação Mieloide/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Nervo Óptico/patologia , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/patologia , Células Ganglionares da Retina/patologia , Transdução de Sinais , Receptor Toll-Like 9/deficiência , Fator de Transcrição Brn-3A/genética , Fator de Transcrição Brn-3A/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Progesterone receptor (PR) is a critical regulator in reproductive tissues that controls a variety of cellular processes. The objective of the study was to study the PR expression in patients with benign prostatic hyperplasia and prostate cancers in connection with the transcription, growth factors, AR, ERα, ERß, and components of the AKT/mTOR signaling pathway expression. MATERIALS AND METHODS: Ninety-seven patients with prostate pathology were enrolled in the study. Forty-two patients had benign prostatic hyperplasia (BH). Fifty-five patients had locally advanced prostate cancer (PCa). The PSA level and the amount of testosterone in the serum were measured using an ELISA assay. The expression level of NF-κB p65, NF-κB p50, HIF-1, HIF-2, growth factor VEGF, VEGFR2, CAIX, as well as AR, ERα, ERß, PR, Brn-3α, TRIM16 were quantified by RT-PCR. The protein level of Brn-3α, TRIM16 was detected by Western Blotting. RESULTS: Growth in PR expression was observed in PCa tissues compared to BH ones without changes in the clinical and pathological features of the patients. An increase in PR expression was detected in patients with PCa compared to BH. Its mRNA level depended on the expression of AR, Brn-3α, and TRIM16, components of the AKT/mTOR signaling pathway, transcription, and growth factors. An increase in the TRIM16 expression in the PCa tissues was noted in the case of a low PR level. We revealed the growth in PR expression was accompanied by the suppression of the signaling cascade activity, AR, Brn-3α mRNA level, and the enhanced PTEN expression in PCa tissues. The increase in PR expression in PCa led to a decrease in the level of mRNA of NF-κB, HIF-1, VEGF, and VEGFR2. CONCLUSION: In general, the data indicated the significance of the PR expression in the development of the prostate pathology that affected the cross-talk between the steroid hormone reception and signal transduction.
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Assuntos
Hiperplasia Prostática/genética , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/metabolismo , Receptores de Progesterona/genética , Serina-Treonina Quinases TOR/genética , Idoso , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Anidrase Carbônica IX/genética , Anidrase Carbônica IX/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Pessoa de Meia-Idade , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Hiperplasia Prostática/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Receptores de Progesterona/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Fator de Transcrição Brn-3A/genética , Fator de Transcrição Brn-3A/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Human embryonic stem cells (hESCs) have the potential to differentiate along the retinal lineage. We have efficiently differentiated human pluripotent stem cells into optic cup-like structures by using a novel retinal differentiation medium (RDM). The purpose of this study was to determine whether the retinal progenitor cells (RPCs) derived from hESCs can integrate into the host retina and differentiate into retinal ganglion cells (RGCs) in vivo. In this study, hESCs (H9-GFP) were induced to differentiate into optic cup-like structures by using our novel differentiation system. The RPCs extracted from the optic cup-like structures were transplanted into the vitreous cavity of N-methyl-d-aspartic acid-treated mice. Sham-treated eyes received the same amount of RDM. The host retinas were analyzed by triple immunofluorescence on the fourth and fifth weeks after transplantation. The optic cup-like structures were efficiently differentiated from hESCs by using our novel differentiation system in vitro for 6-8 weeks. The RPCs extracted from the optic cup-like structures migrated and integrated into the ganglion cell layer (GCL) of the host retina. Furthermore, the remaining transplanted cells were spread over the GCL and had a complementary distribution with host residual RGCs in the GCL of the mouse retina. Surprisingly, some of the transplanted cells expressed the RGC-specific marker Brn3a. These findings demonstrated that the RPCs derived from hESCs could integrate into the host GCL and differentiate into retinal ganglion-like cells in vivo, suggesting that RPCs can be used as an ideal source in supplying countless RGC and embryonic stem cell-based replacement therapies may be a promising treatment to restore vision in patients with degenerative retinal diseases.
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Neurais/transplante , Neurogênese , Células Ganglionares da Retina/citologia , Transplante de Células-Tronco/métodos , Animais , Linhagem Celular , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células Ganglionares da Retina/metabolismo , Fator de Transcrição Brn-3A/genética , Fator de Transcrição Brn-3A/metabolismoRESUMO
We studied reception of sex steroid hormones in the tissues of thyroid papillary cancer and benign tumor. Enhanced expression of AR and ERß mRNA reflected malignant tumor growth. Nuclear factors Brn-3α and TRIM16 modulating expression of steroid hormones play an important role in the development of thyroid tumors. It was found that the level of TRIM16 mRNA is associated with the expression of ERß, which seems to be mediated by its antiestrogen effect.
Assuntos
Proteínas de Ligação a DNA/genética , Receptor beta de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica , Receptores Androgênicos/genética , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Fator de Transcrição Brn-3A/genética , Fatores de Transcrição/genética , Adulto , Idoso , Estudos de Casos e Controles , Proteínas de Ligação a DNA/metabolismo , Receptor beta de Estrogênio/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Androgênicos/metabolismo , Transdução de Sinais , Câncer Papilífero da Tireoide/metabolismo , Câncer Papilífero da Tireoide/patologia , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Fator de Transcrição Brn-3A/metabolismo , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
Excitotoxicity is well known in the neuronal death in the brain and is also linked to neuronal damages in the retina. Recent accumulating evidence show that microglia greatly affect excitotoxicity in the brain, but their roles in retina have received only limited attention. Here, we report that retinal excitotoxicity is mediated by microglia. To this end, we employed three discrete methods, that is, pharmacological inhibition of microglia by minocycline, pharmacological ablation by an antagonist for colony stimulating factor 1 receptor (PLX5622), and genetic ablation of microglia using Iba1-tTA::DTAtetO/tetO mice. Intravitreal injection of NMDA increased the number of apoptotic retinal ganglion cells (RGCs) followed by reduction in the number of RGCs. Although microglia did not respond to NMDA directly, they became reactive earlier than RGC damages. Inhibition or ablation of microglia protected RGCs against NMDA. We found up-regulation of proinflammatory cytokine genes including Il1b, Il6 and Tnfa, among which Tnfa was selectively blocked by minocycline. PLX5622 also suppressed Tnfa expression. Tumor necrosis factor α (TNFα) signals were restricted in microglia at very early followed by spreading into other cell types. TNFα up-regulation in microglia and other cells were significantly attenuated by minocycline and PLX5622, suggesting a central role of microglia for TNFα induction. Both inhibition of TNFα and knockdown of TNF receptor type 1 by siRNA protected RGCs against NMDA. Taken together, our data demonstrate that a phenotypic change of microglia into a neurotoxic one is a critical event for the NMDA-induced degeneration of RGCs, suggesting an importance of non-cell-autonomous mechanism in the retinal neuronal excitotoxicity.
Assuntos
Morte Celular/fisiologia , Citocinas/metabolismo , Microglia/fisiologia , Células Ganglionares da Retina/fisiologia , Aminopiridinas/farmacologia , Animais , Animais Recém-Nascidos , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/genética , Agonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Microglia/ultraestrutura , N-Metilaspartato/farmacologia , Degeneração Neural/induzido quimicamente , Traumatismos do Nervo Óptico/induzido quimicamente , Compostos Orgânicos/farmacologia , Pirróis/farmacologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/ultraestrutura , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição Brn-3A/genética , Fator de Transcrição Brn-3A/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: About 20-30 distinct Retinal Ganglion Cell (RGC) types transmit visual information from the retina to the brain. The developmental mechanisms by which RGCs are specified are still largely unknown. Brn3a is a member of the Brn3/Pou4f transcription factor family, which contains key regulators of RGC postmitotic specification. In particular, Brn3a ablation results in the loss of RGCs with small, thick and dense dendritic arbors ('midget-like' RGCs), and morphological changes in other RGC subpopulations. To identify downstream molecular mechanisms underlying Brn3a effects on RGC numbers and morphology, our group recently performed a RNA deep sequencing screen for Brn3a transcriptional targets in mouse RGCs and identified 180 candidate transcripts. METHODS: We now focus on a subset of 28 candidate genes encoding potential cell type determinant proteins. We validate and further define their retinal expression profile at five postnatal developmental time points between birth and adult stage, using in situ hybridization (ISH), RT-PCR and fluorescent immunodetection (IIF). RESULTS: We find that a majority of candidate genes are enriched in the ganglion cell layer during early stages of postnatal development, but dynamically change their expression profile. We also document transcript-specific expression differences for two example candidates, using RT-PCR and ISH. Brn3a dependency could be confirmed by ISH and IIF only for a fraction of our candidates. CONCLUSIONS: Amongst our candidate Brn3a target genes, a majority demonstrated ganglion cell layer specificity, however only around two thirds showed Brn3a dependency. Some were previously implicated in RGC type specification, while others have known physiological functions in RGCs. Only three genes were found to be consistently regulated by Brn3a throughout postnatal retina development - Mapk10, Tusc5 and Cdh4.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas do Tecido Nervoso/metabolismo , Retina/crescimento & desenvolvimento , Células Ganglionares da Retina/classificação , Células Ganglionares da Retina/metabolismo , Fator de Transcrição Brn-3A/genética , Fatores Etários , Animais , Animais Recém-Nascidos , Caderinas/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , RNA Mensageiro/metabolismo , Retina/citologia , Estatísticas não Paramétricas , Fator de Transcrição Brn-3A/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Purpose: Diabetic retinopathy is a neurovascular disease characterized by increased permeability of the blood-retinal barrier, changes in the neural components of the retina, and low-grade chronic inflammation. Diabetic retinopathy is a major complication of diabetes; however, the impact of a prediabetic state on the retina remains to be elucidated. The aim of this study was to assess possible early retinal changes in prediabetic rats, by evaluating changes in the integrity of the blood-retinal barrier, the retinal structure, neural markers, and inflammatory mediators. Methods: Several parameters were analyzed in the retinas of Wistar rats that drank high sucrose (HSu; 35% sucrose solution during 9 weeks, the prediabetic animal model) and were compared with those of age-matched controls. The permeability of the blood-retinal barrier was assessed with the Evans blue assay, and the content of the tight junction proteins and neural markers with western blotting. Optical coherence tomography was used to evaluate retinal thickness. Cell loss at the ganglion cell layer was assessed with terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay and by evaluating the immunoreactivity of the Brn3a transcription factor. To assess retinal neuroinflammation, the mRNA expression and protein levels of inducible nitric oxide synthase isoform (iNOS), interleukin-1 beta (IL-1ß), and tumor necrosis factor (TNF) were evaluated. Iba1 and MHC-II immunoreactivity and translocator protein (TSPO) mRNA levels were assessed to study the microglial number and activation state. Results: The thickness of the inner retinal layers of the HSu-treated animals decreased. Nevertheless, no apoptotic cells were observed, and no changes in retinal neural markers were detected in the retinas of the HSu-treated animals. No changes were detected in the permeability of the blood-retinal barrier, as well as the tight junction protein content between the HSu-treated rats and the controls. In addition, the inflammatory parameters remained unchanged in the retina despite the tendency for an increase in the number of retinal microglial cells. Conclusions: In a prediabetic rat model, the retinal structure is affected by the thinning of the inner layers, without overt vascular and inflammatory alterations. The results suggest neuronal dysfunction (thinning of the inner retina) that may precede or anticipate the vascular and inflammatory changes. Subtle structural changes might be viewed as early disturbances in an evolving disease, suggesting that preventive strategies (such as the modification of diet habits) could be applied at this stage, before the progression toward irreversible dysfunction and damage to the retina.
Assuntos
Células Ependimogliais/efeitos dos fármacos , Estado Pré-Diabético/diagnóstico , Transdução de Sinais/efeitos dos fármacos , Sacarose/farmacologia , Animais , Barreira Hematorretiniana/efeitos dos fármacos , Permeabilidade Capilar/efeitos dos fármacos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Retinopatia Diabética/induzido quimicamente , Retinopatia Diabética/diagnóstico , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Modelos Animais de Doenças , Células Ependimogliais/citologia , Células Ependimogliais/metabolismo , Azul Evans/química , Regulação da Expressão Gênica , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Estado Pré-Diabético/induzido quimicamente , Estado Pré-Diabético/genética , Estado Pré-Diabético/metabolismo , Ratos , Ratos Wistar , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/ultraestrutura , Tomografia de Coerência Óptica , Fator de Transcrição Brn-3A/genética , Fator de Transcrição Brn-3A/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND/AIMS: The aim of the present study is to investigate the effect of long non-coding RNA-MALAT1 (LncRNA-MALAT1) on retinal ganglion cell (RGC) apoptosis mediated by the PI3K/Akt signaling pathway in rats with glaucoma. METHODS: RGCs were isolated and cultured, and monoclonal antibodies (anti-rat Thy-1, Brn3a and RBPMS) were examined by immunocytochemistry. An overexpression vector MALAT1-RNA activation (RNAa), gene knockout vector MALAT1-RNA interference (RNAi), and control vector MALAT1-negative control (NC) were constructed. A chronic high intraocular pressure (IOP) rat model of glaucoma was established by episcleral vein cauterization. The RGCs were divided into the RGC control, RGC pressure, RGC pressure + MALAT1-NC, RGC pressure + MALAT1-RNAi and RGC pressure + MALAT1-RNAa groups. Sixty Sprague-Dawley (SD) rats were randomly divided into the normal, high IOP, high IOP + MALAT1-NC, high IOP + MALAT1-RNAa and high IOP + MALAT1-RNAi groups. qRT-PCR and western blotting were used to detect the expression levels of LncRNA-MALAT1 and PI3K/Akt. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and flow cytometry were used to detect RGC apoptosis. RESULTS: Immunocytochemistry revealed that the cultured RGCs reached 90% purity. Compared with the RGC pressure + MALAT1-NC group, the RGC pressure + MALAT1-RNAa group exhibited elevated expression levels of MALAT1, lower total protein levels of PI3K and Akt and decreased RGC apoptosis, while these expression levels were reversed in the RGC pressure + MALAT1-RNAi group. RGC numbers and PI3K/Akt expression levels in the high IOP model groups were lower than those in the normal group. In the high IOP + MALAT1-RNAa group, the mRNA and protein expression levels of PI3K/Akt were reduced but higher than those in the other three high IOP model groups. Additionally, RGC numbers in the high IOP + MALAT1-RNAa group were lower than those in the normal group but higher than those in the other three high IOP model groups. CONCLUSION: Our study provides evidence that LncRNA-MALAT1 could inhibit RGC apoptosis in glaucoma through activation of the PI3K/Akt signaling pathway.
Assuntos
Glaucoma/metabolismo , RNA Longo não Codificante/metabolismo , Células Ganglionares da Retina/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Glaucoma/genética , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Pressão Intraocular/genética , Pressão Intraocular/fisiologia , Masculino , Microscopia Eletrônica de Transmissão , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Longo não Codificante/genética , Ratos , Ratos Sprague-Dawley , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo , Fator de Transcrição Brn-3A/genética , Fator de Transcrição Brn-3A/metabolismoRESUMO
Adult neural crest stem-derived cells (NCSC) are of extraordinary high plasticity and promising candidates for use in regenerative medicine. Several locations such as skin, adipose tissue, dental pulp or bone marrow have been described in rodent, as sources of NCSC. However, very little information is available concerning their correspondence in human tissues, and more precisely for human bone marrow. The main objective of this study was therefore to characterize NCSC from adult human bone marrow. In this purpose, we compared human bone marrow stromal cells to human adipose tissue and dermis, already described for containing NCSC. We performed comparative analyses in terms of gene and protein expression as well as functional characterizations. It appeared that human bone marrow, similarly to adipose tissue and dermis, contains NESTIN+ / SOX9+ / TWIST+ / SLUG+ / P75NTR+ / BRN3A+/ MSI1+/ SNAIL1+ cells and were able to differentiate into melanocytes, Schwann cells and neurons. Moreover, when injected into chicken embryos, all those cells were able to migrate and follow endogenous neural crest migration pathways. Altogether, the phenotypic characterization and migration abilities strongly suggest the presence of neural crest-derived cells in human adult bone marrow.
Assuntos
Tecido Adiposo/citologia , Células da Medula Óssea/citologia , Derme/citologia , Células-Tronco Mesenquimais/citologia , Crista Neural/citologia , Células-Tronco Neurais/citologia , Tecido Adiposo/metabolismo , Adulto , Animais , Biomarcadores/metabolismo , Células da Medula Óssea/metabolismo , Diferenciação Celular , Embrião de Galinha , Derme/metabolismo , Feminino , Expressão Gênica , Humanos , Melanócitos/citologia , Melanócitos/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Microinjeções , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina/genética , Nestina/metabolismo , Crista Neural/crescimento & desenvolvimento , Crista Neural/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Células de Schwann/citologia , Células de Schwann/metabolismo , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Fator de Transcrição Brn-3A/genética , Fator de Transcrição Brn-3A/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismoRESUMO
Thalamocortical loops have been implicated in the control of higher-order cognitive functions, but advances in our understanding of the molecular underpinnings of neocortical organization have not been accompanied by similar analyses in the thalamus. Using expression-based correlation maps and the manual mapping of mouse and human datasets available in the Allen Brain Atlas, we identified a few individual regions and several sets of molecularly related nuclei that partially overlap with the classic grouping that is based on topographical localization and thalamocortical connections. These new molecular divisions of the adult thalamic complex are defined by the combinatorial expression of Tcf7l2, Lef1, Gbx2, Prox1, Pou4f1, Esrrg, and Six3 transcription factor genes. Further in silico and experimental analyses provided the evidence that TCF7L2 might be a pan-thalamic specifier. These results provide substantial insights into the "molecular logic" that underlies organization of the thalamic complex.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Núcleos Talâmicos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Atlas como Assunto , Bases de Dados de Compostos Químicos , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Fator de Transcrição Brn-3A/genética , Fator de Transcrição Brn-3A/metabolismo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína Homeobox SIX3RESUMO
The habenula, located on the dorsal thalamic surface, is an emotional and reward processing center. As in the mammalian brain, the zebrafish habenula is divided into dorsal (dHb) and ventral (vHb) subdivisions that project to the interpeduncular nucleus and median raphe (MR) respectively. Previously, we have shown that kisspeptin 1 (Kiss1) expressing in the vHb, regulates the serotonin (5-HT) system in the MR. However, the connectivity between the Kiss1 neurons and the 5-HT system remains unknown. To resolve this issue, we generated a specific antibody against zebrafish Kiss1 receptor (Kiss-R1); using this primary antibody we found intense immunohistochemical labeling in the ventro-anterior corner of the MR (vaMR) but not in 5-HT neurons, suggesting the potential involvement of interneurons in 5-HT modulation by Kiss1. Double-fluorescence labeling showed that the majority of habenular Kiss1 neurons are glutamatergic. In the MR region, Kiss1 fibers were mainly seen in close association with glutamatergic neurons and only scarcely within GABAergic and 5-HT neurons. Our findings indicate that the habenular Kiss1 neurons potentially modulate the 5-HT system primarily through glutamatergic neurotransmission via as yet uncharacterized interneurons. The neuropeptide kisspeptin (Kiss1) play a key role in vertebrate reproduction. We have previously shown modulatory role of habenular Kiss1 in the raphe serotonin (5-HT) systems. This study proposed that the habenular Kiss1 neurons modulate the 5-HT system primarily through glutamatergic neurotransmission, which provides an important insight for understanding of the modulation of 5-HT system by the habenula-raphe pathway.
Assuntos
Ácido Glutâmico/metabolismo , Habenula/citologia , Kisspeptinas/metabolismo , Neurônios/metabolismo , Núcleos da Rafe/citologia , Serotonina/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Glutamato Descarboxilase/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Habenula/metabolismo , Masculino , Dados de Sequência Molecular , Rede Nervosa/metabolismo , RNA Mensageiro/metabolismo , Núcleos da Rafe/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Kisspeptina-1 , Fator de Transcrição Brn-3A/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
The habenular complex in the epithalamus consists of distinct regions with diverse neuronal populations. Past studies have suggested a role for the habenula in voluntary exercise motivation and reinforcement of intracranial self-stimulation but have not assigned these effects to specific habenula subnuclei. Here, we have developed a genetic model in which neurons of the dorsal medial habenula (dMHb) are developmentally eliminated, via tissue-specific deletion of the transcription factor Pou4f1 (Brn3a). Mice with dMHb lesions perform poorly in motivation-based locomotor behaviors, such as voluntary wheel running and the accelerating rotarod, but show only minor abnormalities in gait and balance and exhibit normal levels of basal locomotion. These mice also show deficits in sucrose preference, but not in the forced swim test, two measures of depression-related phenotypes in rodents. We have also used Cre recombinase-mediated expression of channelrhodopsin-2 and halorhodopsin to activate dMHb neurons or silence their output in freely moving mice, respectively. Optical activation of the dMHb in vivo supports intracranial self-stimulation, showing that dMHb activity is intrinsically reinforcing, whereas optical silencing of dMHb outputs is aversive. Together, our findings demonstrate that the dMHb is involved in exercise motivation and the regulation of hedonic state, and is part of an intrinsic reinforcement circuit.
Assuntos
Habenula/fisiologia , Motivação/fisiologia , Atividade Motora/fisiologia , Reforço Psicológico , Animais , Channelrhodopsins , Condicionamento Operante , Preferências Alimentares , Habenula/citologia , Locomoção/genética , Locomoção/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Motivação/genética , Atividade Motora/genética , Neurônios/fisiologia , Optogenética , Autoestimulação , Natação/fisiologia , Sinaptotagminas/genética , Fator de Transcrição Brn-3A/deficiência , Fator de Transcrição Brn-3A/genética , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Although structurally and biochemically similar to the cellular prion (PrP(C)), doppel (Dpl) is unique in its biological functions. There are no reports about any neurodegenerative diseases induced by Dpl. However the artificial expression of Dpl in the PrP-deficient mouse brain causes ataxia with Purkinje cell death. Abundant Dpl proteins have been found in testis and depletion of the Dpl gene (Prnd) causes male infertility. Therefore, we hypothesize different regulations of Prnd in the nerve and male productive systems. In this study, by electrophoretic mobility shift assays we have determined that two different sets of transcription factors are involved in regulation of the Prnd promoter in mouse neuronal N2a and GC-1 spermatogenic (spg) cells, i.e., upstream stimulatory factors (USF) in both cells, Brn-3 and Sp1 in GC-1 spg cells, and Sp3 in N2a cells, leading to the expression of Dpl in GC-1 spg but not in N2a cells. We have further defined that, in N2a cells, Dpl induces oxidative stress and apoptosis, which stimulate ataxia-telangiectasia mutated (ATM)-modulating bindings of transcription factors, p53 and p21, to Prnp promoter, resulting the PrP(C) elevation for counteraction of the Dpl cytotoxicity; in contrast, in GC-1 spg cells, phosphorylation of p21 and N-terminal truncated PrP may play roles in the control of Dpl-induced apoptosis, which may benefit the physiological function of Dpl in the male reproduction system.