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1.
Hum Mol Genet ; 24(25): 7159-70, 2015 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-26376866

RESUMO

DYT6 dystonia is caused by mutations in THAP1 [Thanatos-associated (THAP) domain-containing apoptosis-associated protein] and is autosomal dominant and partially penetrant. Like other genetic primary dystonias, DYT6 patients have no characteristic neuropathology, and mechanisms by which mutations in THAP1 cause dystonia are unknown. Thap1 is a zinc-finger transcription factor, and most pathogenic THAP1 mutations are missense and are located in the DNA-binding domain. There are also nonsense mutations, which act as the equivalent of a null allele because they result in the generation of small mRNA species that are likely rapidly degraded via nonsense-mediated decay. The function of Thap1 in neurons is unknown, but there is a unique, neuronal 50-kDa Thap1 species, and Thap1 levels are auto-regulated on the mRNA level. Herein, we present the first characterization of two mouse models of DYT6, including a pathogenic knockin mutation, C54Y and a null mutation. Alterations in motor behaviors, transcription and brain structure are demonstrated. The projection neurons of the deep cerebellar nuclei are especially altered. Abnormalities vary according to genotype, sex, age and/or brain region, but importantly, overlap with those of other dystonia mouse models. These data highlight the similarities and differences in age- and cell-specific effects of a Thap1 mutation, indicating that the pathophysiology of THAP1 mutations should be assayed at multiple ages and neuronal types and support the notion of final common pathways in the pathophysiology of dystonia arising from disparate mutations.


Assuntos
Cerebelo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Distonia Muscular Deformante/metabolismo , Distonia Muscular Deformante/patologia , Animais , Proteínas de Ligação a DNA/genética , Masculino , Camundongos , Camundongos Mutantes , Mutação , RNA Mensageiro/genética
2.
Front Immunol ; 12: 650522, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33897698

RESUMO

Proinflammatory stimuli lead to endothelial injury, which results in pathologies such as cardiovascular diseases, autoimmune diseases, and contributes to alloimmune responses after organ transplantation. Both mesenchymal stromal cells (MSC) and the extracellular vesicles (EV) released by them are widely studied as regenerative therapy for the endothelium. However, for therapeutic application, the manipulation of living MSC and large-scale production of EV are major challenges. Membrane particles (MP) generated from MSC may be an alternative to the use of whole MSC or EV. MP are nanovesicles artificially generated from the membranes of MSC and possess some of the therapeutic properties of MSC. In the present study we investigated whether MP conserve the beneficial MSC effects on endothelial cell repair processes under inflammatory conditions. MP were generated by hypotonic shock and extrusion of MSC membranes. The average size of MP was 120 nm, and they showed a spherical shape. The effects of two ratios of MP (50,000; 100,000 MP per target cell) on human umbilical vein endothelial cells (HUVEC) were tested in a model of inflammation induced by TNFα. Confocal microscopy and flow cytometry showed that within 24 hours >90% of HUVEC had taken up MP. Moreover, MP ended up in the lysosomes of the HUVEC. In a co-culture system of monocytes and TNFα activated HUVEC, MP did not affect monocyte adherence to HUVEC, but reduced the transmigration of monocytes across the endothelial layer from 138 ± 61 monocytes per microscopic field in TNFα activated HUVEC to 61 ± 45 monocytes. TNFα stimulation induced a 2-fold increase in the permeability of the HUVEC monolayer measured by the translocation of FITC-dextran to the lower compartment of a transwell system. At a dose of 1:100,000 MP significantly decreased endothelial permeability (1.5-fold) respect to TNFα Stimulated HUVEC. Finally, MP enhanced the angiogenic potential of HUVEC in an in vitro Matrigel assay by stimulating the formation of angiogenic structures, such as percentage of covered area, total tube length, total branching points, total loops. In conclusion, MP show regenerative effects on endothelial cells, opening a new avenue for treatment of vascular diseases where inflammatory processes damage the endothelium.


Assuntos
Tecido Adiposo/citologia , Vesículas Extracelulares/imunologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Células-Tronco Mesenquimais/imunologia , Monócitos/imunologia , Adesão Celular/imunologia , Permeabilidade da Membrana Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Microscopia Crioeletrônica , DNA/genética , DNA/isolamento & purificação , Vesículas Extracelulares/genética , Vesículas Extracelulares/ultraestrutura , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Microscopia Eletrônica de Transmissão , Monócitos/citologia , Tamanho da Partícula , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
Front Immunol ; 12: 651109, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33790914

RESUMO

Mesenchymal stromal cells (MSC) are a promising therapy for inflammatory diseases. However, MSC are large and become trapped in the lungs after intravenous infusion, where they have a short survival time. To steer MSC immunoregulatory therapy beyond the lungs, we generated nm-sized particles from MSC membranes (membrane particles, MP), which have immunomodulatory properties, and investigated their internalization and mode of interaction in macrophages subtypes and human umbilical vein endothelial cells (HUVEC) under control and inflammatory conditions. We found that macrophages and HUVEC take up MP in a dose, time, and temperature-dependent manner. Specific inhibitors for endocytotic pathways revealed that MP internalization depends on heparan sulfate proteoglycan-, dynamin-, and clathrin-mediated endocytosis but does not involve caveolin-mediated endocytosis. MP uptake also involved the actin cytoskeleton and phosphoinositide 3-kinase, which are implicated in macropinocytosis and phagocytosis. Anti-inflammatory M2 macrophages take up more MP than pro-inflammatory M1 macrophages. In contrast, inflammatory conditions did not affect the MP uptake by HUVEC. Moreover, MP induced both anti- and pro-inflammatory responses in macrophages and HUVEC by affecting gene expression and cell surface proteins. Our findings on the mechanisms of uptake of MP under different conditions help the development of target-cell specific MP therapy to modulate immune responses.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Micropartículas Derivadas de Células/imunologia , Células-Tronco Mesenquimais/citologia , Micropartículas Derivadas de Células/transplante , Células Cultivadas , Relação Dose-Resposta Imunológica , Voluntários Saudáveis , Células Endoteliais da Veia Umbilical Humana , Humanos , Macrófagos/imunologia , Células-Tronco Mesenquimais/imunologia , Fagocitose/imunologia , Pinocitose/imunologia , Cultura Primária de Células , Gordura Subcutânea/citologia
4.
Stem Cell Res Ther ; 11(1): 462, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33138862

RESUMO

BACKGROUND: Mesenchymal stem cells (MSCs) activate the endogenous immune regulatory system, inducing a therapeutic effect in recipients. MSCs have demonstrated the ability to modulate the differentiation of myeloid cells toward a phagocytic and anti-inflammatory profile. Allogeneic, adipose-derived MSCs (ASCs) have been investigated for the management of complex perianal fistula, with darvadstrocel being the first ASC therapy approved in Europe in March 2018. Additionally, ASCs are being explored as a potential treatment in other indications. Yet, despite these clinical advances, their mechanism of action is only partially understood. METHODS: Freshly isolated human monocytes from the peripheral blood were differentiated in vitro toward M0 non-polarized macrophages (Mphs), M1 pro-inflammatory Mphs, M2 anti-inflammatory Mphs, or mature dendritic cells (mDCs) in the presence or absence of ASCs, in non-contact conditions. The phenotype and function of the differentiated myeloid populations were determined by flow cytometry, and their secretome was analyzed by OLINK technology. We also investigated the capacity of ASCs to modulate the phenotype and function of terminally differentiated M1 Mphs. The role of soluble factors interleukin (IL)-6 and prostaglandin E2 (PGE2) on the ability of ASCs to modulate myeloid cells was assessed using neutralization assays, CRISPR/Cas9 knock-down of cyclooxygenase 2 (COX-2), and ASC-conditioned medium assays using pro-inflammatory stimulus. RESULTS: Co-culture of monocytes in the presence of ASCs resulted in the polarization of Mphs and mDCs toward an anti-inflammatory and phagocytic phenotype. This was characterized by an increase in phagocytic receptors on the cell surface of Mphs (M0, M1, and M2) and mDCs, as well as modulation of chemokine receptors and reduced expression of pro-inflammatory, co-stimulatory molecules. ASCs also modulated the secretome of Mphs and mDCs, demonstrated by reduced expression of pro-inflammatory factors and increased expression of anti-inflammatory and reparative factors. Chemical inhibition of PGE2 with indomethacin abolished this modulatory effect, whereas treatment with a neutralizing anti-IL-6 antibody resulted in a partial abolishment. The knock-down of COX-2 in ASCs and the use of IL-1ß-activated ASC-conditioned media confirmed the key role of PGE2 in ASC-mediated myeloid modulation. In our in vitro experimental settings, ASCs failed to modulate the phenotype and function of terminally polarized M1 Mphs. CONCLUSIONS: The results demonstrate that ASCs are able to modulate the in vitro differentiation of myeloid cells toward an anti-inflammatory and reparative profile. This modulatory effect was mediated mainly by PGE2 and, to a lesser extent, IL-6.


Assuntos
Dinoprostona , Células-Tronco Mesenquimais , Monócitos , Tecido Adiposo , Anti-Inflamatórios , Ciclo-Oxigenase 2 , Humanos , Interleucina-6/genética , Fenótipo
5.
Front Immunol ; 10: 1244, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31258526

RESUMO

Adipose mesenchymal stem cells (ASC) are considered minimally immunogenic. This is due to the low expression of human leukocyte antigens I (HLA-I), lack of HLA-II expression and low expression of co-stimulatory molecules such as CD40 and CD80. The low rate of observed immunological rejection as well as the immunomodulatory qualities, position ASC as a promising cell-based therapy for the treatment of a variety of inflammatory indications. Yet, few studies have addressed relevant aspects of immunogenicity such as ASC donor-to-patient HLA histocompatibility or assessment of immune response triggered by ASC administration, particularly in the cases of presensitization. The present study aims to assess allo-immune responses in a cohort of Crohn's disease patients administered with allogeneic ASC (darvadstrocel formerly Cx601) for the treatment of complex perianal fistulas. We identified donor-specific antibodies (DSA) generation in a proportion of patients and observed that patients showing preexisting immunity were prone to generating DSA after allogeneic therapy. Noteworthy, naïve patients generating DSA at week 12 (W12) showed a significant reduction in DSA titer at week 52 (W52), whereas DSA titer was reduced in pre-sensitized patients only with no specificities against the donor administered. Remarkably, we did not observe any correlation of DSA generation with ASC therapeutic efficacy. In vitro complement-dependent cytotoxicity (CDC) studies have revealed limited cytotoxic levels based upon HLA-I expression and binding capacity even in pro-inflammatory conditions. We sought to identify CDC coping mechanisms contributing to the limited cytotoxic killing observed in ASC in vitro. We found that ASC express membrane-bound complement regulatory proteins (mCRPs) CD55, CD46, and CD59 at basal levels, with CD46 more actively expressed in pro-inflammatory conditions. We demonstrated that CD46 is a main driver of CDC signaling; its depletion significantly enhances sensitivity of ASC to CDC. In summary, despite relatively high clearance, DSA generation may represent a major challenge for allogeneic cell therapy management. Sensitization may be a significant concern when evaluating re-treatment or multi-donor trials. It is still unknown whether DSA generation could potentially be the consequence of donor-to-patient interaction and, therefore, subsequently link to efficacy or biological activity. Lastly, we propose that CDC modulators such as CD46 could be used to ultimately link CDC specificity with allogeneic cell therapy efficacy.


Assuntos
Doença de Crohn/terapia , Fístula/terapia , Rejeição de Enxerto/imunologia , Transplante de Células-Tronco Mesenquimais , Glândulas Perianais/patologia , Complicações Pós-Operatórias/imunologia , Tecido Adiposo/citologia , Adulto , Animais , Células Cultivadas , Estudos de Coortes , Ativação do Complemento , Doença de Crohn/complicações , Feminino , Fístula/complicações , Rejeição de Enxerto/etiologia , Antígenos HLA/imunologia , Humanos , Imunidade Humoral , Imunização , Isoantígenos/imunologia , Masculino , Proteína Cofatora de Membrana/metabolismo , Células-Tronco Mesenquimais/citologia , Glândulas Perianais/cirurgia , Transplante Homólogo
6.
Mol Neurodegener ; 13(1): 33, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29945658

RESUMO

BACKGROUND: Type 2 diabetes (T2D) is a recognized risk factor for the development of cognitive impairment (CI) and/or dementia, although the exact nature of the molecular pathology of T2D-associated CI remains obscure. One link between T2D and CI might involve decreased insulin signaling in brain and/or neurons in either animal or postmortem human brains as has been reported as a feature of Alzheimer's disease (AD). Here we asked if neuronal insulin resistance is a cell autonomous phenomenon in a familial form of AD. METHODS: We have applied a newly developed protocol for deriving human basal forebrain cholinergic neurons (BFCN) from skin fibroblasts via induced pluripotent stem cell (iPSC) technology. We generated wildtype and familial AD mutant PSEN2 N141I (presenilin 2) BFCNs and assessed if insulin signaling, insulin regulation of the major AD proteins Aß and/or tau, and/or calcium fluxes is altered by the PSEN2 N141I mutation. RESULTS: We report herein that wildtype, PSEN2 N141I and CRISPR/Cas9-corrected iPSC-derived BFCNs (and their precursors) show indistinguishable insulin signaling profiles as determined by the phosphorylation of canonical insulin signaling pathway molecules. Chronic insulin treatment of BFCNs of all genotypes led to a reduction in the Aß42/40 ratio. Unexpectedly, we found a CRISPR/Cas9-correctable effect of PSEN2 N141I on calcium flux, which could be prevented by chronic exposure of BFCNs to insulin. CONCLUSIONS: Our studies indicate that the familial AD mutation PSEN2 N141I does not induce neuronal insulin resistance in a cell autonomous fashion. The ability of insulin to correct calcium fluxes and to lower Aß42/40 ratio suggests that insulin acts to oppose an AD-pathophysiology. Hence, our results are consistent with a potential physiological role for insulin as a mediator of resilience by counteracting specific metabolic and molecular features of AD.


Assuntos
Doença de Alzheimer/metabolismo , Neurônios Colinérgicos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Insulina/metabolismo , Doença de Alzheimer/genética , Diabetes Mellitus Tipo 2/complicações , Feminino , Humanos , Resistência à Insulina/fisiologia , Masculino , Mutação , Presenilina-2/genética
7.
Acta Neuropathol Commun ; 5(1): 77, 2017 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-29078805

RESUMO

Basal forebrain cholinergic neurons (BFCNs) are believed to be one of the first cell types to be affected in all forms of AD, and their dysfunction is clinically correlated with impaired short-term memory formation and retrieval. We present an optimized in vitro protocol to generate human BFCNs from iPSCs, using cell lines from presenilin 2 (PSEN2) mutation carriers and controls. As expected, cell lines harboring the PSEN2 N141I mutation displayed an increase in the Aß42/40 in iPSC-derived BFCNs. Neurons derived from PSEN2 N141I lines generated fewer maximum number of spikes in response to a square depolarizing current injection. The height of the first action potential at rheobase current injection was also significantly decreased in PSEN2 N141I BFCNs. CRISPR/Cas9 correction of the PSEN2 point mutation abolished the electrophysiological deficit, restoring both the maximal number of spikes and spike height to the levels recorded in controls. Increased Aß42/40 was also normalized following CRISPR/Cas-mediated correction of the PSEN2 N141I mutation. The genome editing data confirms the robust consistency of mutation-related changes in Aß42/40 ratio while also showing a PSEN2-mutation-related alteration in electrophysiology.


Assuntos
Doença de Alzheimer/fisiopatologia , Sistemas CRISPR-Cas , Neurônios Colinérgicos/fisiologia , Edição de Genes , Células-Tronco Pluripotentes Induzidas/fisiologia , Presenilina-2/genética , Potenciais de Ação , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/metabolismo , Proteínas Reguladoras de Apoptose , Prosencéfalo Basal/metabolismo , Morte Celular , Linhagem Celular , Neurônios Colinérgicos/patologia , Feminino , Heterozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Mutação , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neurogênese , Fragmentos de Peptídeos/metabolismo , Presenilina-2/metabolismo , RNA Mensageiro/metabolismo
8.
Acta Neuropathol Commun ; 2: 139, 2014 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-25231164

RESUMO

Mutations in THAP1 result in dystonia type 6, with partial penetrance and variable phenotype. The goal of this study was to examine the nature and expression pattern of the protein product(s) of the Thap1 transcription factor (DYT6 gene) in mouse neurons, and to study the regional and developmental distribution, and subcellular localization of Thap1 protein. The goal was accomplished via overexpression and knock-down of Thap1 in the HEK293T cell line and in mouse striatal primary cultures and western blotting of embryonic Thap1-null tissue. The endogenous and transduced Thap1 isoforms were characterized using three different commercially available anti-Thap1 antibodies and validated by immunoprecipitation and DNA oligonucleotide affinity chromatography. We identified multiple, novel Thap1 species of apparent Mr 32 kDa, 47 kDa, and 50-52 kDa in vitro and in vivo, and verified the previously identified species at 29-30 kDa in neurons. The Thap1 species at the 50 kDa size range was exclusively detected in murine brain and testes and were located in the nuclear compartment. Thus, in addition to the predicted 25 kDa apparent Mr, we identified Thap1 species with greater apparent Mr that we speculate may be a result of posttranslational modifications. The neural localization of the 50 kDa species and its nuclear compartmentalization suggests that these may be key Thap1 species controlling neuronal gene transcription. Dysfunction of the neuronal 50 kDa species may therefore be implicated in the pathogenesis of DYT6.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Nucléolo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Neurônios/metabolismo , Neurônios/ultraestrutura , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Células Cultivadas , Corpo Estriado/citologia , Embrião de Mamíferos , Regulação da Expressão Gênica/genética , Humanos , Imunoprecipitação , Camundongos , Camundongos Knockout , Chaperonas Moleculares/metabolismo , Peso Molecular , Tecido Nervoso/metabolismo , Tecido Nervoso/patologia , RNA Interferente Pequeno/farmacologia , Ribonucleosídeo Difosfato Redutase , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
9.
PLoS One ; 8(10): e76842, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24204683

RESUMO

Neurotrophins and modifiers of chromatin acetylation and deacetylation participate in regulation of transcription during neuronal maturation and maintenance. The striatal medium spiny neuron is supported by cortically-derived brain derived neurotrophic factor and is the most vulnerable neuron in Huntington's disease, in which growth factor and histone deacetylase activity are both disrupted. We examined the ability of three histone deacetylase inhibitors, trichostatin A, valproic acid and Compound 4 b, alone and combined with brain derived neurotrophic factor (BDNF), to promote phenotypic maturation of striatal medium spiny neurons in vitro. Exposure of these neurons to each of the three compounds led to an increase in overall histone H3 and H4 acetylation, dopamine and cyclic AMP-regulated phosphoprotein, 32 kDa (DARPP-32) mRNA and protein, and mRNA levels of other markers of medium spiny neuron maturation. We were, however, unable to prove that HDAC inhibitors directly lead to remodeling of Ppp1r1b chromatin. In addition, induction of DARPP-32 by brain-derived neurotrophic factor was inhibited by histone deacetylase inhibitors. Although BDNF-induced increases in pTrkB, pAkt, pERK and Egr-1 were unchanged by combined application with VPA, the increase in DARPP-32 was relatively diminished. Strikingly, the NGF1A-binding protein, Nab2, was induced by BDNF, but not in the presence of VPA or TSA. Gel shift analysis showed that α-Nab2 super-shifted a band that is more prominent with extract derived from BDNF-treated neurons than with extracts from cultures treated with VPA alone or VPA plus BDNF. In addition, overexpression of Nab2 induced DARPP-32. We conclude that histone deacetylase inhibitors inhibit the induction of Nab2 by BDNF, and thereby the relative induction of DARPP-32.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Proteínas de Neoplasias/metabolismo , Neurônios/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Acetilação/efeitos dos fármacos , Animais , Western Blotting , Calbindinas/genética , Calbindinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Corpo Estriado/citologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Fosfoproteína 32 Regulada por cAMP e Dopamina/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Histonas/metabolismo , Ácidos Hidroxâmicos/farmacologia , Camundongos , Células NIH 3T3 , Proteínas de Neoplasias/genética , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor trkB/metabolismo , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ácido Valproico/farmacologia
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