Nucleophosmin 1 cooperates with the methyltransferase DOT1L to preserve peri-nucleolar heterochromatin organization by regulating H3K27me3 levels and DNA repeats expression.

BACKGROUND: NPM1 is a phosphoprotein highly abundant in the nucleolus. However, additional nuclear functions have been attributed to NPM1, probably through interaction with other nuclear factors. DOT1L is one interaction partner of NPM1 that catalyzes methylation of histone H3 at lysine 79 (H3K79). DOT1L, playing functional roles in several biological processes, is known for its capability to organize and regulate chromatin. For example, DOT1L modulates DNA repeats expression within peri-nucleolar heterochromatin. NPM1 also affects peri-nucleolar heterochromatin spatial organization. However, it is unclear as of yet whether NPM1 and DOT1L functionally synergize to preserve nucleoli organization and genome stability, and generally, which molecular mechanisms would be involved. RESULTS: We characterized the nuclear function of NPM1 on peri-nucleolar heterochromatin organization. We show that (i) monomeric NPM1 interacts preferentially with DOT1L in the nucleus; (ii) NPM1 acts in concert with DOT1L to maintain each other's protein homeostasis; (iii) NPM1 depletion results in H3K79me2 upregulation and differential enrichment at chromatin binding genes including Ezh2; (iv) NPM1 and DOT1L modulate DNA repeats expression and peri-nucleolar heterochromatin organization via epigenetic mechanisms dependent on H3K27me3. CONCLUSIONS: Our findings give insights into molecular mechanisms employed by NPM1 and DOT1L to regulate heterochromatin activity and structural organization around the nucleoli and shed light on one aspect of the complex role of both proteins in chromatin dynamics.

DOT1L deletion impairs the development of cortical parvalbumin-expressing interneurons.

The cortical plate (CP) is composed of excitatory and inhibitory neurons, the latter of which originate in the ganglionic eminences. From their origin in the ventral telencephalon, maturing postmitotic interneurons migrate during embryonic development over some distance to reach their final destination in the CP. The histone methyltransferase Disruptor of Telomeric Silencing 1-like (DOT1L) is necessary for proper CP development and layer distribution of glutamatergic neurons. However, its specific role on cortical interneuron development has not yet been explored. Here, we demonstrate that DOT1L affects interneuron development in a cell autonomous manner. Deletion of Dot1l in Nkx2.1-expressing interneuron precursor cells results in an overall reduction and altered distribution of GABAergic interneurons in the CP from postnatal day 0 onwards. We observed an altered proportion of GABAergic interneurons in the cortex, with a significant decrease in parvalbumin-expressing interneurons. Moreover, a decreased number of mitotic cells at the embryonic day E14.5 was observed upon Dot1l deletion. Altogether, our results indicate that reduced numbers of cortical interneurons upon DOT1L deletion result from premature cell cycle exit, but effects on postmitotic differentiation, maturation, and migration are likely at play as well.

DOT1L activity affects neural stem cell division mode and reduces differentiation and ASNS expression.

Cortical neurogenesis depends on the balance between self-renewal and differentiation of apical progenitors (APs). Here, we study the epigenetic control of AP's division mode by focusing on the enzymatic activity of the histone methyltransferase DOT1L. Combining lineage tracing with single-cell RNA sequencing of clonally related cells, we show at the cellular level that DOT1L inhibition increases neurogenesis driven by a shift of APs from asymmetric self-renewing to symmetric neurogenic consumptive divisions. At the molecular level, DOT1L activity prevents AP differentiation by promoting transcription of metabolic genes. Mechanistically, DOT1L inhibition reduces activity of an EZH2/PRC2 pathway, converging on increased expression of asparagine synthetase (ASNS), a microcephaly associated gene. Overexpression of ASNS in APs phenocopies DOT1L inhibition, and also increases neuronal differentiation of APs. Our data suggest that DOT1L activity/PRC2 crosstalk controls AP lineage progression by regulating asparagine metabolism.

Human stem cell-based models for studying autism spectrum disorder-related neuronal dysfunction.

The controlled differentiation of pluripotent stem cells (PSCs) into neurons and glia offers a unique opportunity to study early stages of human central nervous system development under controlled conditions in vitro. With the advent of cell reprogramming and the possibility to generate induced pluripotent stem cells (iPSCs) from any individual in a scalable manner, these studies can be extended to a disease- and patient-specific level. Autism spectrum disorder (ASD) is considered a neurodevelopmental disorder, with substantial evidence pointing to early alterations in neurogenesis and network formation as key pathogenic drivers. For that reason, ASD represents an ideal candidate for stem cell-based disease modeling. Here, we provide a concise review on recent advances in the field of human iPSC-based modeling of syndromic and non-syndromic forms of ASD, with a particular focus on studies addressing neuronal dysfunction and altered connectivity. We further discuss recent efforts to translate stem cell-based disease modeling to 3D via brain organoid and cell transplantation approaches, which enable the investigation of disease mechanisms in a tissue-like context. Finally, we describe advanced tools facilitating the assessment of altered neuronal function, comment on the relevance of iPSC-based models for the assessment of pharmaceutical therapies and outline potential future routes in stem cell-based ASD research.

A novel decellularization method to produce brain scaffolds.

Scaffolds composed of extracellular matrix (ECM) can assist tissue remodeling and repair following injury. The ECM is a complex biomaterial composed of proteins, glycoproteins, proteoglycans, and glycosaminoglycans, secreted by cells. The ECM contains fundamental biological cues that modulate cell behavior and serves as a structural scaffold for cell adhesion and growth. For clinical applications, where immune rejection is a constraint, ECM can be processed using decellularization methods intended to remove cells and donor antigens from tissue or organs, while preserving native biological cues essential for cell growth and differentiation. Recent studies show bioengineered organs composed by a combination of a diversity of materials and stem cells as a possibility of new therapeutic strategies to treat diseases that affect different tissues and organs, including the central nervous system (CNS). Nevertheless, the methodologies currently described for brain decellularization involve the use of several chemical reagents with many steps that ultimately limit the process of organ or tissue recellularization. Here, we describe for the first time a fast and straightforward method for complete decellularization of mice brain by the combination of rapid freezing and thawing following the use of only one detergent (Sodium dodecyl sulfate (SDS)). Our data show that using the protocol we describe here, the brain was entirely decellularized, while still maintaining ECM components that are essential for cell survival on the scaffold. Our results also show the cell-loading of the decellularized brain matrix with Neuro2a cells, which were identified by immunohistochemistry in their undifferentiated form. We conclude that this novel and simple method for brain decellularization can be used as a scaffold for cell-loading.

Mechanistic Insights of Astrocyte-Mediated Hyperactive Autophagy and Loss of Motor Neuron Function in SOD1L39R Linked Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with no cure. The reports showed the role of nearby astrocytes around the motor neurons as one among the causes of the disease. However, the exact mechanistic insights are not explored so far. Thus, in the present investigations, we employed the induced pluripotent stem cells (iPSCs) of Cu/Zn-SOD1L39R linked ALS patient to convert them into the motor neurons (MNs) and astrocytes. We report that the higher expression of stress granule (SG) marker protein G3BP1, and its co-localization with the mutated Cu/Zn-SOD1L39R protein in patient's MNs and astrocytes are linked with AIF1-mediated upregulation of caspase 3/7 and hyper activated autophagy. We also observe the astrocyte-mediated non-cell autonomous neurotoxicity on MNs in ALS. The secretome of the patient's iPSC-derived astrocytes exerts significant oxidative stress in MNs. The findings suggest the hyperactive status of autophagy in MNs, as witnessed by the co-distribution of LAMP1, P62 and LC3 I/II with the autolysosomes. Conversely, the secretome of normal astrocytes has shown neuroprotection in patient's iPSC-derived MNs. The whole-cell patch-clamp assay confirms our findings at a physiological functional level in MNs. Perhaps for the first time, we are reporting that the MN degeneration in ALS triggered by the hyper-activation of autophagy and induced apoptosis in both cell-autonomous and non-cell autonomous conditions.

Be Aware of Aggregators in the Search for Potential Human ecto-5'-Nucleotidase Inhibitors.

Promiscuous inhibition due to aggregate formation has been recognized as a major concern in drug discovery campaigns. Here, we report some aggregators identified in a virtual screening (VS) protocol to search for inhibitors of human ecto-5'-nucleotidase (ecto-5'-NT/CD73), a promising target for several diseases and pathophysiological events, including cancer, inflammation and autoimmune diseases. Four compounds (A, B, C and D), selected from the ZINC-11 database, showed IC50 values in the micromolar range, being at the same time computationally predicted as potential aggregators. To confirm if they inhibit human ecto-5'-NT via promiscuous mechanism, forming aggregates, enzymatic assays were done in the presence of 0.01% (v/v) Triton X-100 and an increase in the enzyme concentration by 10-fold. Under both experimental conditions, these four compounds showed a significant decrease in their inhibitory activities. To corroborate these findings, turbidimetric assays were performed, confirming that they form aggregate species. Additionally, aggregation kinetic studies were done by dynamic light scattering (DLS) for compound C. None of the identified aggregators has been previously reported in the literature. For the first time, aggregation and promiscuous inhibition issues were systematically studied and evaluated for compounds selected by VS as potential inhibitors for human ecto-5'-NT. Together, our results reinforce the importance of accounting for potential false-positive hits acting by aggregation in drug discovery campaigns to avoid misleading assay results.

Bradykinin-induced inhibition of proliferation rate during neurosphere differentiation: consequence or cause of neuronal enrichment?

Neural stem cells proliferate and differentiate into neurons and glial cells, being responsible for embryonic and postnatal development of the central nervous system (CNS) as well as for regeneration in the adult brain. These cells also play a key role in maintaining the physiological integrity of the CNS in face of injury or disease. The previous study has demonstrated that bradykinin (BK) treatment simultaneously induces neuronal enrichment (indicating that BK contributes to neurogenesis) and reduced proliferation rates during in vitro differentiation of rat embryonic telencephalon neural precursor cells (NPCs). Here, we provide a mechanism for the unresolved question whether (i) the low rate of proliferation is owed to enhanced neurogenesis or, conversely, (ii) the alteration of the population ratio could result from low proliferation of NPCs and glial cells. In agreement with the previous study, BK promoted neuron-specific ß3-tubulin and MAP2 expression in differentiating embryonic mouse neurospheres, whereas glial protein expression and global proliferation rates decreased. Furthermore, BK augmented the global frequency of cells in G0 -phase of cell cycle after differentiation. Heterogeneous cell populations were observed at this stage, including neurons that always remaining a quiescent state (G0 -phase). It is noteworthy that BK did not interfere with proliferation of any particular cell type, evidenced by coimmunostaining for nestin, ß3-tubulin, glial fibrillary acidic protein (GFAP), and 5-ethynyl-2'-deoxyuridine (EdU). Thus, we conclude that neuronal enrichment is owing only to the fostering of neurogenesis, and that the low proliferation rate on the seventh day of differentiation is a consequence and not the cause of BK-induced neuronal enrichment.

Modulation of mouse embryonic stem cell proliferation and neural differentiation by the P2X7 receptor.

BACKGROUND: Novel developmental functions have been attributed to the P2X7 receptor (P2X7R) including proliferation stimulation and neural differentiation. Mouse embryonic stem cells (ESC), induced with retinoic acid to neural differentiation, closely assemble processes occurring during neuroectodermal development of the early embryo. PRINCIPAL FINDINGS: P2X7R expression together with the pluripotency marker Oct-4 was highest in undifferentiated ESC. In undifferentiated cells, the P2X7R agonist Bz-ATP accelerated cell cycle entry, which was blocked by the specific P2X7R inhibitor KN-62. ESC induced to neural differentiation with retinoic acid, reduced Oct-4 and P2X7R expression. P2X7R receptor-promoted intracellular calcium fluxes were obtained at lower Bz-ATP ligand concentrations in undifferentiated and in neural-differentiated cells compared to other studies. The presence of KN-62 led to increased number of cells expressing SSEA-1, Dcx and ß3-tubulin, as well as the number of SSEA-1 and ß3-tubulin-double-positive cells confirming that onset of neuroectodermal differentiation and neuronal fate determination depends on suppression of P2X7R activity. Moreover, an increase in the number of Ki-67 positive cells in conditions of P2X7R inhibition indicates rescue of progenitors into the cell cycle, augmenting the number of neuroblasts and consequently neurogenesis. CONCLUSIONS: In embryonic cells, P2X7R expression and activity is upregulated, maintaining proliferation, while upon induction to neural differentiation P2X7 receptor expression and activity needs to be suppressed.

Cell cycle regulation during neurogenesis in the embryonic and adult brain.

Throughout the development of the central nervous system, neural crest cells and the primary neural stem cells originate several non-neuronal and neuronal cell types. Undifferentiated stem cells exist in the adult brain, mainly in the dentate gyrus of the hippocampus and in the subventricular zone of the lateral ventricles, and can produce new neurons, participating in brain plasticity and tissue regeneration. Neurogenesis in the embryonic and adult brain occurs under the control of intrinsic and extrinsic factors. However, the mechanisms, by which cell cycle components control neural stem cell proliferation and consequently neurogenesis, still lack further investigation. We discuss here recent knowledge obtained on cell cycle components as key regulators of neural stem and progenitor cell proliferation and differentiation in the embryonic and adult brain.

Functions of neurotrophins and growth factors in neurogenesis and brain repair.

The identification and isolation of multipotent neural stem and progenitor cells in the brain, giving rise to neurons, astrocytes, and oligodendrocytes initiated many studies in order to understand basic mechanisms of endogenous neurogenesis and repair mechanisms of the nervous system and to develop novel therapeutic strategies for cellular regeneration therapies in brain disease. A previous review (Trujillo et al., Cytometry A 2009;75:38-53) focused on the importance of extrinsic factors, especially neurotransmitters, for directing migration and neurogenesis in the developing and adult brain. Here, we extend our review discussing the effects of the principal growth and neurotrophic factors as well as their intracellular signal transduction on neurogenesis, fate determination and neuroprotective mechanisms. Many of these mechanisms have been elucidated by in vitro studies for which neural stem cells were isolated, grown as neurospheres, induced to neural differentiation under desired experimental conditions, and analyzed for embryonic, progenitor, and neural marker expression by flow and imaging cytometry techniques. The better understanding of neural stem cells proliferation and differentiation is crucial for any therapeutic intervention aiming at neural stem cell transplantation and recruitment of endogenous repair mechanisms.

Identificação de resíduos de treonina e tirosina importantes na regulação da atividade do receptor P2X4 humano através de mutagênese sítio-dirigida / Identification of threonine and tyrosine residues important for human P2X4 receptor activity by site-directed mutagenesis

O receptor P2X4 (canal iônico controlado por adenosina-5'-trifosfato-ATP) está amplamente distribuído no sistema nervoso central e, após sua ativação, pode regular os níveis de cálcio intracelulares via permeação direta e por ativação de canais de cálcio voltagem-dependentes. Tem sido proposto que a atividade do receptor pode ser importante na plasticidade sináptica. Tendo em vista a importância do receptor P2X4, sobretudo na fisiologia do sistema nervoso central, é útil caracterizá-lo farmacologicamente e entender os mecanismos moleculares que regulam sua atividade. Examinamos o papel que resíduos específicos N- e C-terminais desempenham na atividade do receptor P2X4 humano, combinando técnicas de biologia molecular, bioquímica e patch-clamp em células de rim de embrião humano (células HEK-293T). Células HEK-293T expressando o receptor P2X4 wild-type apresentaram correntes iônicas, cujas amplitudes dependeram da concentração de ATP, fornecendo um valor de EC50 de 1,37 ± 0,21 µM. Os receptores mutantes E14A e D16A exibiram respostas ao ATP equiparáveis àquelas do receptor selvagem, ao passo que os mutantes Y15A e T17A não foram funcionais, apesar de serem expressos na membrana plasmática das células. A inibição de tirosina fosfatases por pervanadato diminuiu fortemente correntes induzidas por ATP. Subsequente análise de citometria de fluxo na presença de um anticorpo contra resíduos de fosfotirosina indicaram que, entre as células que expressam o receptor P2X4, a percentagem de células fosfo-tirosina-positivas é a mesma para os mutantes Y372A (86 ± 10%) e Y378A (79 ± 6.9%), mas substancialmente menor para os mutantes Y15A (35 ± 12%), Y367A (48 ± 6.4%) e Y372F (31 ± 1.7%), quando comparados com células que expressam o receptor wild-type (76 ± 5.6%). Resultados semelhantes foram obtidos quando quantificamos a expressão relativa de proteínas fosforiladas em resíduos de tirosina e expressamos através dos valores de intensidade de fluorescência média. Ensaios de western-blot revelaram que mesmo o mutante T17A é fosforilado em resíduos de treonina, sugerindo que o receptor P2X4 contém outros sítios de fosforilação. Entretanto, nenhum sinal de fosfotirosina foi detectado no receptor wild-type e nos mutantes, em que resíduos de tirosina foram substituídos por alanina ou fenilalanina. Não parece ser o resíduo Y15 o alvo de tal fosforilação, cabendo a ele um papel estrutural mais importante. Nossos dados também sugerem que a fosforilação em resíduos de tirosina de proteínas intermediárias regula a atividade do receptor P2X4

The human P2X4 receptor (ATP-gated ion channel) is widely distributed in the CNS and, after activation, participates in regulation of levels of intracellular calcium through direct permeation and activation of voltage-dependent calcium channels with well-defined functions including synaptic plasticity. Given the importance of the P2X4 receptor, especially in CNS physiology, we investigated the role that specific N- and C-termini residues play in human P2X4 receptor activity, by combining techniques of molecular biology, biochemistry and patch-clamping in human embryonic kidney cells (HEK-293T cells). HEK-293T cells expressing the wild-type P2X4 receptor showed ionic currents whose amplitudes depended on the ATP concentration, providing an EC50 value of 1.37 ± 0.21 mM. E14A and D16A receptor mutants exhibited responses to ATP comparable to those ones of wild-type receptor, whereas Y15A and T17A mutants were not functional, despite being expressed in the plasma membrane of cells. The inhibition of tyrosine phosphatases by pervanadate decreased strongly ATP-induced currents. Subsequent flow cytometry analysis in the presence of an antibody against phosphotyrosine residues indicated that, among the cells that express the P2X4 receptor, the percentage of phosphotyrosine-positive cells was the same for Y372A (86 ± 10%) and Y378A (79 ± 6.9%) mutants, however, substantially lower for Y15A (35 ± 12%), Y367A (48 ± 6.4%) and Y372F (31 ± 1.7%) mutants when compared with cells expressing the wild-type receptor (76 ± 5.6%). Similar results were obtained by quantifying the relative expression of phosphotyrosine proteins. Western blot assays revealed that even the T17A mutant was phosphorylated at threonine residues, suggesting that the human P2X4 receptor also contains further phosphorylation sites. However, no phosphotyrosine-antibody signal was detected in the wild-type receptor and mutants in which tyrosine residues were replaced by alanine or phenylalanine. The residue Y15 is supposedly not the target of such phosphorylation, despite its important structural role. However, the present work indicates that tyrosine phosphorylation of intermediate signaling proteins regulates P2X4 receptor activity

Lipophilicity as a determinant of binding of procaine analogs to rat α3ß4 nicotinic acetylcholine receptor.

Nicotinic acetylcholine receptors (nAChRs) have been studied in detail with regard to their interaction with therapeutic and drug addiction-related compounds. Using a structure-activity approach, we have examined the relationship among the molecular features of a set of eight para-R-substituted N,N-[(dimethylamino)ethyl] benzoate hydrochlorides, structurally related to procaine and their affinity for the α(3)ß(4) nAChR heterologously expressed in KXα3ß4R2 cells. Affinity values (log[1/IC50]) of these compounds for the α(3)ß(4) nAChR were determined by their competition with [(3)H]TCP binding. Log(1/IC50) values were analyzed considering different hydrophobic and electronic parameters and those related to molar refractivity. These have been experimentally determined or were taken from published literature. In accordance with literature observations, the generated cross-validated quantitative structure-activity relationship (QSAR) equations indicated a significant contribution of hydrophobic term to binding affinity of procaine analogs to the receptor and predicted affinity values for several local anesthetics (LAs) sets taken from the literature. The predicted values by using the QSAR model correlated well with the published values both for neuronal and for electroplaque nAChRs. Our work also reveals the general structure features of LAs that are important for interaction with nAChRs as well as the structural modifications that could be made to enhance binding affinity.

Inhibition mechanism of rat α3ß4 nicotinic acetylcholine receptor by the Alzheimer therapeutic tacrine.

Nicotinic acetylcholine receptors (nAChRs) were studied in detail in the past regarding their interaction with therapeutic and drug addiction related compounds. Using fast kinetic whole-cell recording, we have now studied effects of tacrine, an agent used clinically to treat Alzheimer's disease, on currents elicited by activation of rat α(3)ß(4) nAChR heterologously expressed in KXα3ß4R2 cells. Characterization of receptor activation by nicotine used as agonist revealed a K(d) of 23 ± 0.2 µM and 4.3 ± 1.3 for the channel opening equilibrium constant, Φ(-1). Experiments were performed to investigate whether tacrine is able to activate the α(3)ß(4) nAChR. Tacrine did not activate whole-cell currents in KXα3ß4R2 cells but inhibited receptor activity at submicromolar concentration. Dose-response curves obtained with increasing agonist or inhibitor concentration revealed competitive inhibition of nAChRs by tacrine, with an apparent inhibition constant, K(I), of 0.8 µM. The increase of Φ(-1) in the presence of tacrine suggests that the drug stabilizes a nonconducting open channel form of the receptor. Binding studies with TCP and MK-801 ruled out tacrine binding to common allosteric sites of the receptor. Our study suggests a novel mechanism for action of tacrine on nAChRs besides inhibition of acetylcholine esterase.