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- PD-1 and ICOS are coexpressed in T follicular helper cells but define three stages of maturation of T follicular regulatory cellsPublication . Ribeiro, Filipa; Antunes, Diogo; Pires, Ana R.; Rino, José; Filipe, Beatriz; Jesus, Kátia; Correia, Ricardo; Fonseca, Válter R; Kumar, Saumya; Graca, LuisHumoral responses to infection or vaccination require T cell-B cell interactions. T follicular helper (TFH) cells drive germinal center (GC) responses by providing help to B cells, whereas T follicular regulatory (TFR) cells regulate them. Both mature GC-located TFH and TFR cells are widely characterized by the expression of ICOS and PD-1. However, although human TFR cells share many phenotypic characteristics with TFH cells, we found that ICOS and PD-1 are up-regulated differently in each. Although TFH cells express these proteins synchronously during maturation, they define three maturation stages in TFR cells. TFR cells in an intermediate maturation stage express ICOS, and it is only at the last stage of differentiation that both molecules are expressed at high levels. Although most TFR cells within the B cell follicle are PD-1-, the TFR within the GC are ICOS+PD-1+. These results show that TFH and TFR cells within human lymphoid tissue follow distinct maturation stages.
- Pharmacological inhibition of the spliceosome subunit SF3b triggers EJC-independent NMDPublication . Carvalho, Teresa; Martins, Sandra; Rino, José; Marinho, Sérgio; Carmo-Fonseca, MariaSpliceostatin A, meayamycin, and pladienolide B are small molecules that target the SF3b subunit of the spliceosomal U2 small nuclear ribonucleoprotein (snRNP). These compounds are attracting much attention as tools to manipulate splicing and for use as potential anti-cancer drugs. We investigated the effects of these inhibitors on mRNA transport and stability in human cells. Upon splicing inhibition, unspliced pre-mRNAs accumulated in the nucleus, particularly within enlarged nuclear speckles. However, a small fraction of the pre-mRNA molecules were exported to the cytoplasm. We identified the export adaptor ALYREF as being associated with intron-containing transcripts and show its requirement for the nucleo-cytoplasmic transport of unspliced pre-mRNA. In contrast, the exon junction complex (EJC) core protein eIF4AIII failed to form a stable complex with intron-containing transcripts. Despite the absence of EJC, unspliced transcripts in the cytoplasm were degraded by nonsense-mediated decay (NMD), suggesting that unspliced transcripts are degraded by an EJC-independent NMD pathway. Collectively, our results indicate that although blocking the function of SF3b elicits a massive accumulation of unspliced pre-mRNAs in the nucleus, intron-containing transcripts can still bind the ALYREF export factor and be transported to the cytoplasm, where they trigger an alternative NMD pathway.
- Adenosine A2A receptors modulate α-Synuclein aggregation and toxicityPublication . Ferreira, Diana G.; Batalha, Vânia; Vicente Miranda, Hugo; Coelho, Joana E; Gomes, Rui; Gonçalves, Francisco Q.; Real, Joana I.; Rino, José; Albino-Teixeira, António; Cunha, Rodrigo A.; Outeiro, Tiago; Lopes, Luisa V.Abnormal accumulation of aggregated α-synuclein (aSyn) is a hallmark of sporadic and familial Parkinson's disease (PD) and related synucleinopathies. Recent studies suggest a neuroprotective role of adenosine A2A receptor (A2AR) antagonists in PD. Nevertheless, the precise molecular mechanisms underlying this neuroprotection remain unclear. We assessed the impact of A2AR blockade or genetic deletion (A2AR KO) on synaptic plasticity and neuronal cell death induced by aSyn oligomers. We found that impairment of LTP associated with aSyn exposure was rescued in A2AR KO mice or upon A2AR blockade, through an NMDA receptor-dependent mechanism. The mechanisms underlying these effects were evaluated in SH-SY5Y cells overexpressing aSyn and rat primary neuronal cultures exposed to aSyn. Cell death in both conditions was prevented by selective A2AR antagonists. Interestingly, blockade of these receptors did not interfere with aSyn oligomerization but, instead, reduced the percentage of cells displaying aSyn inclusions. Altogether, our data raise the possibility that the well-documented effects of A2AR antagonists involve the control of the latter stages of aSyn aggregation, thereby preventing the associated neurotoxicity. These findings suggest that A2AR represent an important target for the development of effective drugs for the treatment of PD and related synucleinopathies.
- Dynamics and interactions of nuclear proteins revealed by quantitative photobleaching microscopyPublication . Rino, José; Fonseca, M. Carmo, 1959-; Soares, Eduardo Ducla, 1944-The nucleus is a complex cellular organelle, exhibiting a high degree of organization and also a highly dynamic nature. Live cell imaging using fluorescent proteins (FPs) as molecular tags and photobleaching techniques have been essential in revealing the dynamic nature of the cell nucleus. In this thesis, these tools were used to study molecular dynamics and interactions inside this cellular compartment. Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) were used to analyze the kinetic behavior of spliceosome components SmE, U2AF65, U2AF35, SF1 and SC35 in the nucleus of living cells. The recruitment mechanism of splicing factors (SFs) to the sites of transcription is still poorly understood. Our results rule out the hypothesis that a transcription specific signal recruits SFs from the speckles. They also suggest the formation of multi-protein complexes distinct from the spliceosome. The existence of these complexes was confirmed by Fluorescence Resonance Energy Transfer (FRET) techniques, which revealed that SFs could interact with each other even in the absence of active splicing. A novel U2AF65 self-interaction was also detected, suggesting altogether that levels of SFs in speckles are consistent with self-organization mechanisms. The intranuclear mobility of mRNPs was studied using two GFP-tagged mRNA-binding proteins, PABPN1 and TAP, as mRNA markers. A novel FLIP method was devised to quantify the mobility of the RNA-bound and unbound pools of molecules and used to test whether myosin motors were implicated in mRNP movement. We show that this is not the case and that myosin inhibition appears to affect transcription instead. A novel FLIP after Photoactivation method was developed to study the nucleocytoplasmic exchange dynamics of nuclear proteins, yielding the permanence times of molecules inside the nucleus. The method was used to study the role of the structural domains of TAP in its shuttling activity.
- Decrease of CD68 synovial macrophages in celastrol treated arthritic ratsPublication . Cascao, Rita; Vidal, Bruno; Lopes, Inês; Paisana, Eunice; Rino, José; Moita, Luis; Fonseca, João EuricoBackground: Rheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease characterized by cellular infiltration into the joints, hyperproliferation of synovial cells and bone damage. Available treatments for RA only induce remission in around 30% of the patients, have important adverse effects and its use is limited by their high cost. Therefore, compounds that can control arthritis, with an acceptable safety profile and low production costs are still an unmet need. We have shown, in vitro, that celastrol inhibits both IL-1β and TNF, which play an important role in RA, and, in vivo, that celastrol has significant anti-inflammatory properties. Our main goal in this work was to test the effect of celastrol in the number of sublining CD68 macrophages (a biomarker of therapeutic response for novel RA treatments) and on the overall synovial tissue cellularity and joint structure in the adjuvant-induced rat model of arthritis (AIA). Methods: Celastrol was administered to AIA rats both in the early (4 days after disease induction) and late (11 days after disease induction) phases of arthritis development. The inflammatory score, ankle perimeter and body weight were evaluated during treatment period. Rats were sacrificed after 22 days of disease progression and blood, internal organs and paw samples were collected for toxicological blood parameters and serum proinflammatory cytokine quantification, as well as histopathological and immunohistochemical evaluation, respectively. Results: Here we report that celastrol significantly decreases the number of sublining CD68 macrophages and the overall synovial inflammatory cellularity, and halted joint destruction without side effects. Conclusions: Our results validate celastrol as a promising compound for the treatment of arthritis.
- Live-cell visualization of pre-mRNA splicing with single-molecule sensitivityPublication . Martin, Robert M.; Rino, José; Carvalho, Célia; Kirchhausen, Tomas; Carmo-Fonseca, MariaRemoval of introns from pre-messenger RNAs (pre-mRNAs) via splicing provides a versatile means of genetic regulation that is often disrupted in human diseases. To decipher how splicing occurs in real time, we directly examined with single-molecule sensitivity the kinetics of intron excision from pre-mRNA in the nucleus of living human cells. By using two different RNA labeling methods, MS2 and λN, we show that β-globin introns are transcribed and excised in 20-30 s. Furthermore, we show that replacing the weak polypyrimidine (Py) tract in mouse immunoglobulin μ (IgM) pre-mRNA by a U-rich Py decreases the intron lifetime, thus providing direct evidence that splice-site strength influences splicing kinetics. We also found that RNA polymerase II transcribes at elongation rates ranging between 3 and 6 kb min(-1) and that transcription can be rate limiting for splicing. These results have important implications for a mechanistic understanding of cotranscriptional splicing regulation in the live-cell context.
- Frontiers in fluorescence microscopyPublication . Rino, José; Braga, José; Henriques, Ricardo; Carmo-Fonseca, MariaHow we see organisms and cells depends on the tools at our disposal. For over 150 years, biologists were forced to rely on fixed, dehydrated and stained specimens in order to guess how the living cells could function. It all changed abruptly during the last two decades when the rapid development of novel imaging techniques revolutionized the way scientists look at the structures of life alive.
- Depletion of the yeast nuclear exosome subunit Rrp6 results in accumulation of Polyadenylated RNAs in a discrete domain within the nucleolusPublication . Carneiro, Tiago; Carvalho, Célia; Braga, José; Rino, José; Milligan, Laura; Tollervey, David; Carmo-Fonseca, MariaRecent data reveal that a substantial fraction of transcripts generated by RNA polymerases I, II, and III are rapidly degraded in the nucleus by the combined action of the exosome and a noncanonical poly(A) polymerase activity. This work identifies a domain within the yeast nucleolus that is enriched in polyadenylated RNAs in the absence of the nuclear exosome RNase Rrp6 or the exosome cofactor Mtr4. In normal yeast cells, poly(A)(+) RNA was undetectable in the nucleolus but the depletion of either Rrp6 or Mtr4 led to the accumulation of polyadenylated RNAs in a discrete subnucleolar region. This nucleolar poly(A) domain is enriched for the U14 snoRNA and the snoRNP protein Nop1 but is distinct from the nucleolar body that functions in snoRNA maturation. In strains lacking both Rrp6 and the poly(A) polymerase Trf4, the accumulation of poly(A)(+) RNA was suppressed, suggesting the involvement of the Trf4-Air1/2-Mtr4 polyadenylation (TRAMP) complex. The accumulation of polyadenylated snoRNAs in a discrete nucleolar domain may promote their recognition as substrates for the exosome.
- RNA seeds nuclear bodiesPublication . Carmo-Fonseca, Maria; Rino, JoséThe interior of the eukaryotic cell nucleus is populated by a multitude of microscopic domains termed nuclear bodies. Despite having attracted much attention, how these compartments form and are maintained remained elusive. Now, two live-cell imaging studies provide compelling evidence that nascent RNAs can act as transiently immobilized scaffolds that recruit specific nuclear body proteins.
- A new protocol for whole-brain biodistribution analysis of AAVs by tissue clearing, light-sheet microscopy and semi-automated spatial quantificationPublication . Lopes, Miguel M.; Paysan, Jacques; Rino, José; Lopes, Sara M.; Pereira de Almeida, Luís; Cortes, Luísa; Nobre, Rui JorgeRecombinant adeno-associated virus (rAAV) has become one of the most promising gene delivery systems for both in vitro and in vivo applications. However, a key challenge is the lack of suitable imaging technologies to evaluate delivery, biodistribution and tropism of rAAVs and efficiently monitor disease amelioration promoted by AAV-based therapies at a whole-organ level with single-cell resolution. Therefore, we aimed to establish a new pipeline for the biodistribution analysis of natural and new variants of AAVs at a whole-brain level by tissue clearing and light-sheet fluorescence microscopy (LSFM). To test this platform, neonatal C57BL/6 mice were intravenously injected with rAAV9 encoding EGFP and, after sacrifice, brains were processed by standard immunohistochemistry and a recently released aqueous-based clearing procedure. This clearing technique required no dedicated equipment and rendered highly cleared brains, while simultaneously preserving endogenous fluorescence. Moreover, three-dimensional imaging by LSFM allowed the quantitative analysis of EGFP at a whole-brain level, as well as the reconstruction of Purkinje cells for the retrieval of valuable morphological information inaccessible by standard immunohistochemistry. In conclusion, the pipeline herein described takes the AAVs to a new level when coupled to LSFM, proving its worth as a bioimaging tool in tropism and gene therapy studies.