Katia Carneiro

Synphilin-1 Is Developmentally Localized to Synaptic Terminals, and Its Association with Synaptic Vesicles Is Modulated by α-Synuclein

α-Synuclein is the major component of Lewy bodies in patients with Parkinsons disease, and mutations in the α-synuclein gene are responsible for some familial forms of the disease. α-Synuclein is enriched in the presynapse, but its synaptic targets are unknown. Synphilin-1 associates in vivo with α-synuclein promoting the formation of intracellular inclusions. Additionally synphilin-1 has been found to be an intrinsic component of Lewy bodies in patients with Parkinsons disease. To understand the role of synphilin-1 in Parkinsons disease, we sought to define its localization and function in the brain. We now report that, like α-synuclein, synphilin-1 was enriched in neurons. In young rats, synphilin-1 was prominent in neuronal cell bodies but gradually migrated to neuropil during development. Immunoelectron microscopy of adult rat cerebral cortex demonstrated that synphilin-1 was highly enriched in presynaptic nerve terminals. Synphilin-1 co-immunoprecipitated with synaptic vesicles, indicating a strong association with these structures. In vitro binding experiments demonstrated that the N terminus of synphilin-1 robustly associated with synaptic vesicles and that this association was resistant to high salt washing but was abolished by inclusion of α-synuclein in the incubation medium. Our data indicated that synphilin-1 is a synaptic partner of α-synuclein, and it may mediate synaptic roles attributed to α-synuclein.

Histone Deacetylase activity is necessary for left-right patterning during vertebrate development

BackgroundConsistent asymmetry of the left-right (LR) axis is a crucial aspect of vertebrate embryogenesis. Asymmetric gene expression of the TGFβ superfamily member Nodal related 1 (Nr1) in the left lateral mesoderm plate is a highly conserved step regulating the situs of the heart and viscera. In Xenopus, movement of maternal serotonin (5HT) through gap-junctional paths at cleavage stages dictates asymmetry upstream of Nr1. However, the mechanisms linking earlier biophysical asymmetries with this transcriptional control point are not known.ResultsTo understand how an early physiological gradient is transduced into a late, stable pattern of Nr1 expression we investigated epigenetic regulation during LR patterning. Embryos injected with mRNA encoding a dominant-negative of Histone Deacetylase (HDAC) lacked Nr1 expression and exhibited randomized sidedness of the heart and viscera (heterotaxia) at stage 45. Timing analysis using pharmacological blockade of HDACs implicated cleavage stages as the active period. Inhibition during these early stages was correlated with an absence of Nr1 expression at stage 21, high levels of heterotaxia at stage 45, and the deposition of the epigenetic marker H3K4me2 on the Nr1 gene. To link the epigenetic machinery to the 5HT signaling pathway, we performed a high-throughput proteomic screen for novel cytoplasmic 5HT partners associated with the epigenetic machinery. The data identified the known HDAC partner protein Mad3 as a 5HT-binding regulator. While Mad3 overexpression led to an absence of Nr1 transcription and randomized the LR axis, a mutant form of Mad3 lacking 5HT binding sites was not able to induce heterotaxia, showing that Mad3s biological activity is dependent on 5HT binding.ConclusionHDAC activity is a new LR determinant controlling the epigenetic state of Nr1 from early developmental stages. The HDAC binding partner Mad3 may be a new serotonin-dependent regulator of asymmetry linking early physiological asymmetries to stable changes in gene expression during organogenesis.

The ATP-sensitive K+-channel (KATP) controls early left–right patterning in Xenopus and chick embryos

Consistent left-right asymmetry requires specific ion currents. We characterize a novel laterality determinant in Xenopus laevis: the ATP-sensitive K(+)-channel (K(ATP)). Expression of specific dominant-negative mutants of the Xenopus Kir6.1 pore subunit of the K(ATP) channel induced randomization of asymmetric organ positioning. Spatio-temporally controlled loss-of-function experiments revealed that the K(ATP) channel functions asymmetrically in LR patterning during very early cleavage stages, and also symmetrically during the early blastula stages, a period when heretofore largely unknown events transmit LR patterning cues. Blocking K(ATP) channel activity randomizes the expression of the left-sided transcription of Nodal. Immunofluorescence analysis revealed that XKir6.1 is localized to basal membranes on the blastocoel roof and cell-cell junctions. A tight junction integrity assay showed that K(ATP) channels are required for proper tight junction function in early Xenopus embryos. We also present evidence that this function may be conserved to the chick, as inhibition of K(ATP) in the primitive streak of chick embryos randomizes the expression of the left-sided gene Sonic hedgehog. We propose a model by which K(ATP) channels control LR patterning via regulation of tight junctions.

HDAC Activity Is Required during Xenopus Tail Regeneration

The ability to fully restore damaged or lost organs is present in only a subset of animals. The Xenopus tadpole tail is a complex appendage, containing epidermis, muscle, nerves, spinal cord, and vasculature, which regenerates after amputation. Understanding the mechanisms of tail regeneration may lead to new insights to promote biomedical regeneration in non-regenerative tissues. Although chromatin remodeling is known to be critical for stem cell pluripotency, its role in complex organ regeneration in vivo remains largely uncharacterized. Here we show that histone deacetylase (HDAC) activity is required for the early stages of tail regeneration. HDAC1 is expressed during the 1st two days of regeneration. Pharmacological blockade of HDACs using Trichostatin A (TSA) increased histone acetylation levels in the amputated tail. Furthermore, treatment with TSA or another HDAC inhibitor, valproic acid, specifically inhibited regeneration. Over-expression of wild-type Mad3, a transcriptional repressor known to associate in a complex with HDACs via Sin3, inhibited regeneration. Similarly, expression of a Mad3 mutant lacking the Sin3-interacting domain that is required for HDAC binding also blocks regeneration, suggesting that HDAC and Mad3 may act together to regulate regeneration. Inhibition of HDAC function resulted in aberrant expression of Notch1 and BMP2, two genes known to be required for tail regeneration. Our results identify a novel early role for HDAC in appendage regeneration and suggest that modulation of histone acetylation is important in regenerative repair of complex appendages.

Epigenetic Control of Macrophage Shape Transition towards an Atypical Elongated Phenotype by Histone Deacetylase Activity

Inflammatory chronic pathologies are complex processes characterized by an imbalance between the resolution of the inflammatory phase and the establishment of tissue repair. The main players in these inflammatory pathologies are bone marrow derived monocytes (BMDMs). However, how monocyte differentiation is modulated to give rise to specific macrophage subpopulations (M1 or M2) that may either maintain the chronic inflammatory process or lead to wound healing is still unclear. Considering that inhibitors of Histone Deacetylase (HDAC) have an anti-inflammatory activity, we asked whether this enzyme would play a role on monocyte differentiation into M1 or M2 phenotype and in the cell shape transition that follows. We then induced murine bone marrow progenitors into monocyte/macrophage differentiation pathway using media containing GM-CSF and the HDAC blocker, Trichostatin A (TSA). We found that the pharmacological inhibition of HDAC activity led to a shape transition from the typical macrophage pancake-like shape into an elongated morphology, which was correlated to a mixed M1/M2 profile of cytokine and chemokine secretion. Our results present, for the first time, that HDAC activity acts as a regulator of macrophage differentiation in the absence of lymphocyte stimuli. We propose that HDAC activity down regulates macrophage plasticity favoring the pro-inflammatory phenotype.

The Ca2+-dependent protease Calpain A regulates Cactus/IκB levels during Drosophila development in response to maternal Dpp signals

Regulation of NF kappaB activity is central to many processes during development and disease. Activation of NF kappaB family members depends on degradation of inhibitory I kappaB proteins. In Drosophila, a nuclear gradient of the NF kappaB/c-rel protein Dorsal subdivides the embryonic dorsal-ventral axis, defining the extent and location of mesodermal and ectodermal territories. Activation of the Toll pathway directs Dorsal nuclear translocation by inducing proteosomal degradation of the I kappaB homologue Cactus. Another mechanism that impacts on Dorsal activation involves the Toll-independent pathway, which regulates constitutive Cactus degradation. We have shown that the BMP protein Decapentaplegic (Dpp) inhibits Cactus degradation independent of Toll. Here we report on a novel element of this pathway: the calcium-dependent protease Calpain A. Calpain A knockdowns increase Cactus levels, shifting the Dorsal gradient and dorsal-ventral patterning. As shown for mammalian I kappaB, this effect requires PEST sequences in the Cactus C-terminus, implying a conserved role for calpains. Alteration of Calpain A or dpp results in similar effects on Dorsal target genes. Epistatic analysis confirms Calpain A activity is regulated by Dpp, indicating that Dpp signals increase Cactus levels through Calpain A inhibition, thereby interfering with Dorsal activation. This mechanism may allow coordination of Toll, BMP and Ca(2+) signals, conferring precision to Dorsal-target expression domains.

Nitrergic dendrites in the superficial layers of the rat superior colliculus: Retinal afferents and alternatively spliced isoforms in normal and deafferented animals

The superficial layers of the rat superior colliculus (sSC) receive innervation from the retina and include nitrergic neurons. We have shown previously that in sSC, eye enucleation reduces NADPH diaphorase staining considerably in all but the most proximal dendrites of nitrergic neurons. We have used immunocytochemistry for neuronal nitric oxide synthase (nNOS) at light and electron microscopic levels and bilateral eye enucleation with varied survival times to determine the regulatory changes imposed by the direct and indirect loss of retinal input on apparent nNOS amount and subcellular distribution. In addition, we have used SDS‐PAGE and immunoblotting to test alternatively spliced isoforms in normal and deafferented animals. Our results show that unambiguously identified retinal terminals contact nitrergic neurons. In normal dendrites, nNOS immunoreactivity was distributed almost completely within the cytoplasm of the dendrite and along the postsynaptic membrane at synaptic junctions, in association with endoplasmic reticulum, ribosomes and external mitochondrial membranes. In contrast, nNOS labeling was greatly reduced in sSC deprived of retinal projections, and could only be observed in association with mitochondrial membranes and postsynaptic densities. Immunoblots of the soluble fraction from sSC revealed a surprisingly high proportion of the β isoform with respect to the α counterpart in normal colliculi, suggesting an increase in isoform proportion after enucleation, or at least maintenance of the same proportion. It is suggested that ultrastructural alterations observed in sSC cells of enucleated animals may be consequent to plastic reactions of the sSC cells in response to the removal of retinal afferents.

A critical survey on nitric oxide synthase expression and nitric oxide function in the retinotectal system

The ongoing research on the roles of the gas nitric oxide (NO) in the nervous system has demonstrated its involvement in neurotransmission, synaptic plasticity, learning, excitotoxicity, neurodegenerative diseases and regulation of the cerebral blood flow. Thus, this molecule has been currently considered an important neuromodulator in CNS. Studies carried out in the visual system, particularly in the retinotectal component, have contributed to this current concept about NO. In the present work, we reviewed critically current data about nitric oxide synthase (NOS) expression in the superior colliculus/optic tectum, as well as the roles of NO in the formation of the retinotopic map and in synaptic plasticity. Several vertebrate species have been used in studies about the NOS expression in the retinotectal system and most of the available results are in agreement with the involvement of NO in the developmental refinement of the retinotectal projections, and its role as a neuromodulator of synaptic function during the processing of visual information. However, the few studies about the functional linkage between NOS expression/NO synthesis and retinotectal topographic refinement/tectal synaptic plasticity are not conclusive and/or sometimes inconsistent, indicating that more experimental data are necessary to improve the understanding about NO functions in this visual subsystem. Predictive models for the involvement of NO as a retrograde messenger in the developmental retinotectal refinement are discussed.

Galectin-3, histone deacetylases, and Hedgehog signaling: Possible convergent targets in schistosomiasis-induced liver fibrosis

Schistosomiasis affects approximately 240 million people in the world. Schistosoma mansoni eggs in the liver induce periportal fibrosis and hepatic failure driven by monocyte recruitment and macrophage activation, resulting in robust Th2 response. Here, we suggested a possible involvement of Galectin-3 (Gal-3), histone deacetylases (HDACs), and Hedgehog (Hh) signaling with macrophage activation during Th1/Th2 immune responses, fibrogranuloma reaction, and tissue repair during schistosomiasis. Gal-3 is highly expressed by liver macrophages (Kupffer cells) around Schistosoma eggs. HDACs and Hh regulate macrophage polarization and hepatic stellate cell activation during schistosomiasis-associated fibrogenesis. Previously, we demonstrated an abnormal extracellular matrix distribution in the liver that correlated with atypical monocyte–macrophage differentiation in S. mansoni-infected, Gal-3-deficient (Lgals3-/-) mice. New findings explored in this review focus on the chronic phase, when wild-type (Lgals3+/+) and Lgals3-/- mice were analyzed 90 days after cercariae infection. In Lgals3-/- infected mice, there was significant inflammatory infiltration with myeloid cells associated with egg destruction (hematoxylin and eosin staining), phagocytes (specifically Kupffer cells), numerically reduced and diffuse matrix extracellular deposition in fibrotic areas (Gomori trichrome staining), and severe disorganization of collagen fibers surrounding the S. mansoni eggs (reticulin staining). Granuloma-derived stromal cells (GR cells) of Lgals3-/- infected mice expressed lower levels of alpha smooth muscle actin (α-SMA) and eotaxin and higher levels of IL-4 than Lgals3+/+ mice (real-time PCR). The relevant participation of macrophages in these events led us to suggest distinct mechanisms of activation that culminate in defective fibrosis in the liver of Lgals3-/- infected mice. These aspects were discussed in this review, as well as the possible interference between Gal-3, HDACs, and Hh signaling during progressive liver fibrosis in S. mansoni-infected mice. Further studies focused on macrophage roles could elucidate these questions and clear the potential utility of these molecules as antifibrotic targets.

Chick embryo xenograft model reveals a novel perineural niche for human adipose-derived stromal cells

ABSTRACT Human adipose-derived stromal cells (hADSC) are a heterogeneous cell population that contains adult multipotent stem cells. Although it is well established that hADSC have skeletal potential in vivo in adult organisms, in vitro assays suggest further differentiation capacity, such as into glia. Thus, we propose that grafting hADSC into the embryo can provide them with a much more instructive microenvironment, allowing the human cells to adopt diverse fates or niches. Here, hADSC spheroids were grafted into either the presumptive presomitic mesoderm or the first branchial arch (BA1) regions of chick embryos. Cells were identified without previous manipulations via human-specific Alu probes, which allows efficient long-term tracing of heterogeneous primary cultures. When grafted into the trunk, in contrast to previous studies, hADSC were not found in chondrogenic or osteogenic territories up to E8. Surprisingly, 82.5% of the hADSC were associated with HNK1+ tissues, such as peripheral nerves. Human skin fibroblasts showed a smaller tropism for nerves. In line with other studies, hADSC also adopted perivascular locations. When grafted into the presumptive BA1, 74.6% of the cells were in the outflow tract, the final goal of cardiac neural crest cells, and were also associated with peripheral nerves. This is the first study showing that hADSC could adopt a perineural niche in vivo and were able to recognize cues for neural crest cell migration of the host. Therefore, we propose that xenografts of human cells into chick embryos can reveal novel behaviors of heterogeneous cell populations, such as response to migration cues.