Wpływ kwasu retinowego na komórki pheochromocytoma

Introduction: Retinoic acid is a regulator of gene expression which, by binding to its nuclear receptor, determines the degree of differentiation in multiple cancer cell types. On the basis of this capability it was introduced, e.g. in the therapy of neuroblastoma. In cells derived from neural crest, such as neuroblastoma cells, retinoic acid initiates differentiation into neurons. This substance acts in a similar way on a rat pheochromocytoma cell line PC12. The aim of our work was to examine the influence of retinoic acid on the phenotype of human pheochromocytoma cells in primary culture. Material and methods: Observations were made on two primary cultures isolated from human pheochromocytoma. Cells were grown in RPMI1640 medium supplemented with 10% foetal bovine serum. Subsequently, the cultures were treated with 100 mMol retinoic acid for three-days. An evaluation of the phenotype change was performed by estimating the expression levels of F-actin, MAP-2 protein, and chromogranin, with the use of a confocal microscopy. Results: The introduction of retinoic acid into the culture caused an increase in the F-actin level and its redistribution in the form of stress fibers. Simultaneously, the cells changed their shape, generating more processes. No change was detected in the expression level of neuroendocrine markers: MAP-2 and chromogranin. Conclusions: Retinoic acid appears to have an influence on some phenotype parameters of human pheochromocytoma cells. Further work is needed to determine the molecular mechanisms of this process, and to evaluate thoroughly the benefits of introducing retinoic acid into therapy of pheochromocytoma tumors.Wstęp: Kwas retinowy jest regulatorem ekspresji genów, który działając poprzez receptor jądrowy, wpływa na stopień zróżnicowania wielu typów komórek nowotworowych. Właściwości te stały się podstawą zastosowania go w onkologii, m.in. w terapii neuroblastoma. W komórkach pochodzących z grzebienia nerwowego, takich jak komórki neuroblastoma, kwas retinowy uruchamia proces różnicowania w kierunku neuronów. Substancja ta działa w sposób zbliżony również na komórki szczurzej linii pheochromocytoma (PC12). Celem pracy było zbadanie wpływu kwasu retinowego na cechy fenotypowe ludzkich komórek pheochromocytoma w warunkach hodowli pierwotnej. Materiał i metody: Badania przeprowadzono na dwóch hodowlach pierwotnych wyprowadzonych z ludzkich guzów pheochromocytoma. Komórki hodowano w pożywce RPMI1640 z dodatkiem 10-procentowej surowicy bydlęcej, a następnie poddane działaniu kwasu retinowego, w stężeniu 100 mM przez 3 dni. Oceny fenotypu dokonano poprzez badanie ekspresji F-aktyny, białka MAP-2 (microtubule-associated protein-2, marker neuronalny), i chromograniny (marker neuroendokrynny), z detekcją przy u¿yciu mikroskopu konfokalnego. Wyniki: Działanie kwasem retinowym spowodowało zwiększenie zawartości F-aktyny i jej redystrybucję w formie włókien naprężeniowych, co znalazło odzwierciedlenie w zmianie kształtu komórek, które utworzyły więcej wypustek. Nie stwierdzono zmian w poziomie ekspresji markerów neuroendokrynnych: MAP-2 i chromograniny. Wnioski: Kwas retinowy prawdopodobnie wpływa na niektóre parametry fenotypowe ludzkich komórek pheochromocytoma. Konieczne jest przeprowadzenie dalszych badań w celu wyjaśnienia mechanizmów molekularnych tego zjawiska oraz głębszej oceny perspektyw zastosowania kwasu retinowego w leczeniu guza chromochłonnego

Ultrastructural visualization of 3D chromatin folding using volume electron microscopy and DNA in situ hybridization.

The human genome is extensively folded into 3-dimensional organization. However, the detailed 3D chromatin folding structures have not been fully visualized due to the lack of robust and ultra-resolution imaging capability. Here, we report the development of an electron microscopy method that combines serial block-face scanning electron microscopy with in situ hybridization (3D-EMISH) to visualize 3D chromatin folding at targeted genomic regions with ultra-resolution (5 × 5 × 30 nm in xyz dimensions) that is superior to the current super-resolution by fluorescence light microscopy. We apply 3D-EMISH to human lymphoblastoid cells at a 1.7 Mb segment of the genome and visualize a large number of distinctive 3D chromatin folding structures in ultra-resolution. We further quantitatively characterize the reconstituted chromatin folding structures by identifying sub-domains, and uncover a high level heterogeneity of chromatin folding ultrastructures in individual nuclei, suggestive of extensive dynamic fluidity in 3D chromatin states

Vascular cognitive impairment linked to brain endothelium inflammation in early stages of heart failure in mice

Background Although advanced heart failure ( HF ) is a clinically documented risk factor for vascular cognitive impairment, the occurrence and pathomechanisms of vascular cognitive impairment in early stages of HF are equivocal. Here, we characterize vascular cognitive impairment in the early stages of HF development and assess whether cerebral hypoperfusion or prothrombotic conditions are involved. Methods and Results Tgαq*44 mice with slowly developing isolated HF triggered by cardiomyocyte‐specific overexpression of G‐αq*44 protein were studied before the end‐stage HF , at the ages of 3, 6, and 10 months: before left ventricle dysfunction; at the stage of early left ventricle diastolic dysfunction (with preserved ejection fraction); and left ventricle diastolic/systolic dysfunction, respectively. In 6‐ to 10‐month‐old but not in 3‐month‐old Tgαq*44 mice, behavioral and cognitive impairment was identified with compromised blood‐brain barrier permeability, most significantly in brain cortex, that was associated with myelin sheet loss and changes in astrocytes and microglia. Brain endothelial cells displayed increased E‐selectin immunoreactivity, which was accompanied by increased amyloid‐β 1‐42 accumulation in piriform cortex and increased cortical oxidative stress (8‐ OH dG immunoreactivity). Resting cerebral blood flow measured by magnetic resonance imaging in vivo was preserved, but ex vivo NO ‐dependent cortical arteriole flow regulation was impaired. Platelet hyperreactivity was present in 3‐ to 10‐month‐old Tgαq*44 mice, but it was not associated with increased platelet‐dependent thrombogenicity. Conclusions We report for the first time that vascular cognitive impairment is already present in the early stage of HF development, even before left ventricle systolic dysfunction. The underlying pathomechanism, independent of brain hypoperfusion, involves preceding platelet hyperreactivity and brain endothelium inflammatory activation. </jats:sec

KAT3-dependent acetylation of cell type-specific genes maintains neuronal identity in the adult mouse brain.

The lysine acetyltransferases type 3 (KAT3) family members CBP and p300 are important transcriptional co-activators, but their specific functions in adult post-mitotic neurons remain unclear. Here, we show that the combined elimination of both proteins in forebrain excitatory neurons of adult mice resulted in a rapidly progressing neurological phenotype associated with severe ataxia, dendritic retraction and reduced electrical activity. At the molecular level, we observed the downregulation of neuronal genes, as well as decreased H3K27 acetylation and pro-neural transcription factor binding at the promoters and enhancers of canonical neuronal genes. The combined deletion of CBP and p300 in hippocampal neurons resulted in the rapid loss of neuronal molecular identity without de- or transdifferentiation. Restoring CBP expression or lysine acetylation rescued neuronal-specific transcription in cultured neurons. Together, these experiments show that KAT3 proteins maintain the excitatory neuron identity through the regulation of histone acetylation at cell type-specific promoter and enhancer regions.The ultrastructure research was sup- ported by the Polish National Science Center Grant UMO-2014/15/N/NZ3/04468 and by the European Regional Development Fund POIG 01.01.02-00-008/08. J.P.L.-A. research is supported by Grants RYC-2015-18056 and RTI2018-102260-B-I00 from MICINN co- financed by ERDF. A.B. research is supported by Grants SAF2017-87928-R, PCIN-2015- 192-C02-01, and SEV-2017-0723 from MICINN co-financed by ERDF, PROMETEO/ 2016/026 from the Generalitat Valenciana, and RGP0039/2017 from the Human Fron- tiers Science Program Organization (HFSPO). The Instituto de Neurociencias is a “Centre of Excellence Severo Ochoa”

Activation-induced chromatin reorganization in neurons depends on HDAC1 activity

Spatial chromatin organization is crucial for transcriptional regulation and might be particularly important in neurons since they dramatically change their transcriptome in response to external stimuli. We show that stimulation of neurons causes condensation of large chromatin domains. This phenomenon can be observed in vitro in cultured rat hippocampal neurons as well as in vivo in the amygdala and hippocampal neurons. Activity-induced chromatin condensation is an active, rapid, energy-dependent, and reversible process. It involves calcium-dependent pathways but is independent of active transcription. It is accompanied by the redistribution of posttranslational histone modifications and rearrangements in the spatial organization of chromosome territories. Moreover, it leads to the reorganization of nuclear speckles and active domains located in their proximity. Finally, we find that the histone deacetylase HDAC1 is the key regulator of this process. Our results suggest that HDAC1-dependent chromatin reorganization constitutes an important level of transcriptional regulation in neurons.This work was supported by the National Science Centre grant nos. UMO-2015/18/E/NZ3/00730 (A.M., A.G., H.S.N., E.J. and Y.Y.), 2014/15/N/NZ2/00379 and 2017/24/T/NZ2/00307 (P.T.), 2019/35/O/ST6/02484 (D.P. and G.B.), and 2014/14/M/NZ4/00561 (K.H.O. and R.K.F.). B.W. and B.G. were supported by the Foundation for Polish Science TEAM-TECH Core Facility project “NGS platform for comprehensive diagnostics and personalized therapy in neuro-oncology,” Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund (TEAM to D.P.). A.M.G. was supported by the H2020-MSCA-COFUND-2014 grant Bio4Med (GA no. 665735).Peer reviewe

Fine-structural distribution of MMP-2 and MMP-9 activities in the rat skeletal muscle upon training: a study by high-resolution in situ zymography

Matrix metalloproteinases (MMPs) are key regulators of extracellular matrix remodeling, but have also important intracellular targets. The purpose of this study was to examine the activity and subcellular localization of the gelatinases MMP-2 and MMP-9 in skeletal muscle of control and physically trained rats. In control hind limb muscle, the activity of the gelatinases was barely detectable. In contrast, after 5 days of intense exercise, in Soleus (Sol), but not Extensor digitorum longus (EDL) muscle, significant upregulation of gelatinolytic activity in myofibers was observed mainly in the nuclei, as assessed by high resolution in situ zymography. The nuclei of quiescent satellite cells did not contain the activity. Within the myonuclei, the gelatinolytic activity colocalized with an activated RNA Polymerase II. Also in Sol, but not in EDL, there were few foci of mononuclear cells with strongly positive cytoplasm, associated with apparent necrotic myofibers. These cells were identified as activated satellite cells/myoblasts. No extracellular gelatinase activity was observed. Gel zymography combined with subcellular fractionation revealed training-related upregulation of active MMP-2 in the nuclear fraction, and increase of active MMP-9 in the cytoplasmic fraction of Sol. Using RT-PCR, selective increase in MMP-9 mRNA was observed. We conclude that training activates nuclear MMP-2, and increases expression and activity of cytoplasmic MMP-9 in Sol, but not in EDL. Our results suggest that the gelatinases are involved in muscle adaptation to training, and that MMP-2 may play a novel role in myonuclear functions

Thoracic Hemisection in Rats Results in Initial Recovery Followed by a Late Decrement in Locomotor Movements, with Changes in Coordination Correlated with Serotonergic Innervation of the Ventral Horn

<div><p>Lateral thoracic hemisection of the rodent spinal cord is a popular model of spinal cord injury, in which the effects of various treatments, designed to encourage locomotor recovery, are tested. Nevertheless, there are still inconsistencies in the literature concerning the details of spontaneous locomotor recovery after such lesions, and there is a lack of data concerning the quality of locomotion over a long time span after the lesion. In this study, we aimed to address some of these issues. In our experiments, locomotor recovery was assessed using EMG and CatWalk recordings and analysis. Our results showed that after hemisection there was paralysis in both hindlimbs, followed by a substantial recovery of locomotor movements, but even at the peak of recovery, which occurred about 4 weeks after the lesion, some deficits of locomotion remained present. The parameters that were abnormal included abduction, interlimb coordination and speed of locomotion. Locomotor performance was stable for several weeks, but about 3–4 months after hemisection secondary locomotor impairment was observed with changes in parameters, such as speed of locomotion, interlimb coordination, base of hindlimb support, hindlimb abduction and relative foot print distance. Histological analysis of serotonergic innervation at the lumbar ventral horn below hemisection revealed a limited restoration of serotonergic fibers on the ipsilateral side of the spinal cord, while on the contralateral side of the spinal cord it returned to normal. In addition, the length of these fibers on both sides of the spinal cord correlated with inter- and intralimb coordination. In contrast to data reported in the literature, our results show there is not full locomotor recovery after spinal cord hemisection. Secondary deterioration of certain locomotor functions occurs with time in hemisected rats, and locomotor recovery appears partly associated with reinnervation of spinal circuitry by serotonergic fibers.</p></div

Comparison of EMG activity of the forelimb and hindlimb muscles.

<p>EMG activity during locomotion of a representative rat before (A), 7 days (B), 14 days (C) and 42 days (D) after spinal cord hemisection. Interlimb coordination (onset of EMG activity in the left TA (l TA) with respect to the contralateral right TA (r TA)) and intralimb coordination (onset of EMG activity in the left extensor (l Sol) with respect to the left TA (l TA)) for these time points are shown in the left and right polar plots, respectively. Zero corresponds to the onset of activity in the right TA muscle, and the positions of the filled black circles indicate the times of onset of activity in the left TA<i>—</i>interlimb coordination (left panel) or the onset of activity in the left Sol <i>-</i>intralimb coordination (right panel) in the relation to the onset of activity in the left TA muscle. The black lines each represent data from one animal, and the red line represents the global mean for the group. l Tri—left <i>Triceps</i>; l/r Sol—left and right <i>Soleus</i> and l/r TA—left and right <i>Tibialis Anterior</i>.</p

DP-b99 modulates matrix metalloproteinase activity and neuronal plasticity.

DP-b99 is a membrane-activated chelator of zinc and calcium ions, recently proposed as a therapeutic agent. Matrix metalloproteinases (MMPs) are zinc-dependent extracellularly operating proteases that might contribute to synaptic plasticity, learning and memory under physiological conditions. In excessive amounts these enzymes contribute to a number of neuronal pathologies ranging from the stroke to neurodegeneration and epileptogenesis. In the present study, we report that DP-b99 delays onset and severity of PTZ-induced seizures in mice, as well as displays neuroprotective effect on kainate excitotoxicity in hippocampal organotypic slices and furthermore blocks morphological reorganization of the dendritic spines evoked by a major neuronal MMP, MMP-9. Taken together, our findings suggest that DP-b99 may inhibit neuronal plasticity driven by MMPs, in particular MMP-9, and thus may be considered as a therapeutic agent under conditions of aberrant plasticity, such as those subserving epileptogenesis