Reyna Hernández-Benítez

In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration

Targeted genome editing via engineered nucleases is an exciting area of biomedical research and holds potential for clinical applications. Despite rapid advances in the field, in vivo targeted transgene integration is still infeasible because current tools are inefficient, especially for non-dividing cells, which compose most adult tissues. This poses a barrier for uncovering fundamental biological principles and developing treatments for a broad range of genetic disorders. Based on clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) technology, here we devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more importantly, in vivo (for example, in neurons of postnatal mammals). As a proof of concept of its therapeutic potential, we demonstrate the efficacy of HITI in improving visual function using a rat model of the retinal degeneration condition retinitis pigmentosa. The HITI method presented here establishes new avenues for basic research and targeted gene therapies.

Thrombin potently enhances swelling-sensitive glutamate efflux from cultured astrocytes

High concentrations of thrombin (Thr) have been linked to neuronal damage in cerebral ischemia and traumatic brain injury. In the present study we found that Thr markedly enhanced swelling‐activated efflux of 3H‐glutamate from cultured astrocytes exposed to hyposmotic medium. Thr (0.5–5 U/mL) elicited small 3H‐glutamate efflux under isosmotic conditions and increased the hyposmotic glutamate efflux by 5‐ to 10‐fold, the maximum effect being observed at 15% osmolarity reduction. These Thr effects involve its protease activity and are fully mimicked by SFFLRN, the synthetic peptide activating protease‐activated receptor‐1. Thr potentiation of 3H‐glutamate efflux was largely dependent on a Thr‐elicited increases in cytosolic Ca2+ (Ca2+i) concentration ([Ca2+]i). Preventing Ca2+i rise by treatment with EGTA‐AM or with the phospholipase C blocker U73122 reduced the Thr‐increased glutamate efflux by 68%. The protein kinase C blockers Go6976 or chelerythrine reduced the Thr effect by 19%–22%, while Ca/calmodulin blocker W7 caused a 63% inhibition. In addition to this Ca2+‐sensitive pathway, Thr effect on glutamate efflux also involved activation of phosphoinositide‐3 kinase (PI3K), since it was reduced by the PI3K inhibitor wortmannin (51% inhibition). Treating cells with EGTA‐AM plus wortmannin essentially abolished Thr‐dependent glutamate efflux. Thr‐activated glutamate release was potently inhibited by the blockers of the volume‐sensitive anion permeability pathway, NPPB (IC50 15.8 μM), DCPIB (IC50 4.2 μM). These results suggest that Thr may contribute to the excitotoxic neuronal injury by elevating extracellular glutamate release from glial cells. Therefore, this work may aid in search of neuroprotective strategies for treating cerebral ischemia and brain trauma.

Taurine stimulates proliferation of mice embryonic cultured neural progenitor cells

Taurine is present in high levels in fetal brain which decrease in the adult, suggesting its role in brain development. In some regions of taurine deficient animals cells show defective migration and the presence of numerous mitotic figures, suggesting a delay in cell proliferation. To know more about the role of taurine in the developing brain cells, the present study investigated whether taurine is a factor involved in proliferation or/and viability of neural progenitor cells (NPC). NPC were obtained from 13.5‐days mice embryos mesencephalon, and cultured during 4–5 days to form neurospheres in the presence of EGF plus FGFb (EGF/FGF) or EGF alone. Mesencephalon taurine content (349 mmoles/kg protein) was lost in NPC and recovered after addition of 10 mM taurine to the culture. Neurospheres‐forming NPC were over 94% nestin‐positive. Taurine increased 38.6% and 43.2% the number of NPC formed in EGF/FGF or EGF conditions, respectively. In secondary neurospheres this increase was 24.6% and 62.1%, in EGF/FGF or EGF cultures respectively. Correspondingly neurospheres size was increased by taurine but neurospheres number was not enhanced. Taurine significantly increased the number of BrdU‐positive cells, without affecting cell viability, suggesting proliferation as the mechanism responsible for taurine action increasing NPC. Taurine seems unable to increase the number of β‐III‐tubulin‐positive cells differentiated from neurospheres after serum addition, and rather an increase in astrocytes was observed. These results point to taurine as a trophic factor contributing to optimize NPC proliferation.

Taurine stimulates proliferation and promotes neurogenesis of mouse adult cultured neural stem/progenitor cells.

This study reports an effect of taurine (1-10 mM) increasing markedly (120%) the number of neural precursor cells (NPCs) from adult mouse subventricular zone, cultured as neurospheres. This effect is one of the highest reported for adult neural precursor cells. Taurine-containing cultures showed 73-120% more cells than controls, after 24 and 96 h in culture, respectively. Taurine effect is due to enhanced proliferation as assessed by BrdU incorporation assays. In taurine cultures BrdU incorporation was markedly higher than controls from 1.5 to 48 h, with the maximal difference found at 1.5 h. This effect of taurine reproduced at every passage with the same window time. Taurine effects are not mimicked by glycine, alanine or GABA. Clonal efficiency values of 3.6% for taurine cultures and 1.3% for control cultures suggest a taurine influence on both, progenitor and stem cells. Upon differentiation, the proportion of neurons in control and taurine cultures was 3.1% (±0.5) and 10.2% (±0.8), respectively. These results are relevant for taurine implication in brain development as well as in adult neurogenesis. Possible mechanisms underlying taurine effects on cell proliferation are discussed.

On the Role of G-Protein Coupled Receptors in Cell Volume Regulation

Cell volume is determined genetically for each cell lineage, but it is not a static feature of the cell. Intracellular volume is continuously challenged by metabolic reactions, uptake of nutrients, intracellular displacement of molecules and organelles and generation of ionic gradients. Moreover, recent evidence raises the intriguing possibility that changes in cell volume act as signals for basic cell functions such as proliferation, migration, secretion and apoptosis. Cells adapt to volume increase by a complex, dynamic process resulting from the concerted action of volume sensing mechanisms and intricate signaling chains, directed to initiate the multiple adaptations demanded by a change in cell volume, among others adhesion reactions, membrane and cytoskeleton remodeling, and activation of the osmolyte pathways leading to restablish the water balance between extracellular/intracellular or intracellular/intracellular compartments. In multicellular organisms, a continuous interaction with the external milieu is fundamental for the dynamics of the cell. It is in this sense that the recent surge of interest about the influence on cell volume control by the most extended family of signaling elements, the G proteins, acquires particular importance. As here reviewed, a large variety of G-protein coupled receptors (GPCRs) are involved in this interplay with cell volume regulatory mechanisms, which amplifies and diversifies the volume-elicited signaling chains, providing a variety of routes towards the multiple effectors related to cell volume changes.

Taurine enhances the growth of neural precursors derived from fetal human brain and promotes neuronal specification.

Taurine is present at high concentrations in the fetal brain and is required for optimal brain development. Recent studies have reported that taurine causes increased proliferation of neural stem/progenitor neural cells (neural precursor cells, NPCs) obtained from embryonic and adult rodent brain. The present study is the first to show that taurine markedly increases cell numbers in cultures and neuronal generation from human NPCs (hNPCs). hNPCs obtained from 3 fetal brains (14-15 weeks of gestation) were cultured and expanded as neurospheres, which contained 76.3% nestin-positive cells. Taurine (5-20 mM) increased the number of hNPCs in culture, with maximal effect found at 10 mM and 4 days of culture. The taurine-induced increase ranged from 57 to 188% in the 3 brains examined. Taurine significantly enhanced the percentage of neurons formed from hNPCs under differentiating conditions, with increases ranging from 172 to 480% over controls without taurine. Taurine also increased the cell number and neuronal generation in cultures of the immortalized human cell line ReNcell VM. These results suggest that taurine has a positive influence on hNPC growth and neuronal formation.

Multiple mechanisms mediate the taurine-induced proliferation of neural stem/progenitor cells from the subventricular zone of the adult mouse.

Taurine was previously reported to increase the proliferation of neural precursor cells (NPCs) from subventricular zone of the mouse brain. The results of a study that aimed to understand the mechanisms of this effect are presented here. Because taurine was not found in NPC nuclei, direct interactions with nuclear elements seem unlikely. A gene expression profile analysis indicated that genes that are regulated by taurine have roles in i) proliferation, including the Shh and Wnt pathways; ii) cellular adhesion; iii) cell survival; and iv) mitochondrial functioning. Cell cycle analysis of propidium iodide and CFSE-labeled cells using flow cytometry revealed an increase in the number of cells in the S-phase and a decrease in those in the G0/G1 phase in taurine-treated cultures. No changes in the length of the cell cycle were observed. Quantification of the viable, apoptotic, and necrotic cells in cultures using flow cytometry and calcein-AM, annexin-V, and propidium iodide staining showed reductions in the number of apoptotic and necrotic cells (18% to 11% and 13% to 10%, respectively) and increases in the number of viable cells (61% to 69%) in the taurine-treated cultures. Examination of the relative mitochondrial potential values by flow cytometry and rhodamine123 or JC-1 staining showed a 44% increase in the number of cells with higher mitochondrial potential and a 38% increase in the mitochondrial membrane potential in taurine cultures compared with those of controls. Taken together, the results suggest that taurine provides more favorable conditions for cell proliferation by improving mitochondrial functioning.

Taurine and brain development: trophic or cytoprotective actions?

The decline of taurine content during brain maturation as well as the consequences of taurine deficiency disturbing brain development, suggest its involvement in basic processes of developing brain cells. If taurine participates in cell protection, differentiation or proliferation in the developing brain is as yet unclear. Extensive and solid evidence supports taurine cytoprotective actions, directly or indirectly related to an antioxidant effect. Since redox status and oxidative stress are now implicated in signalling processes regulating cell differentiation and proliferation, the question is raised of whether the taurine antioxidant activity is on the basis of its requirement during brain development.

Thrombin potentiates d‐aspartate efflux from cultured astrocytes under conditions of K+ homeostasis disruption

Thrombin levels increase in brain during ischemia and hemorrhagic episodes, and may contribute to excitotoxic neural damage. This study examined the effect of thrombin on glutamate efflux from rat cortical cultured astrocytes using 3H‐d‐aspartate as radiotracer. The glutamate efflux was initiated by addition of 100 mM K+ plus 1 mM ouabain (K/O) to replicate extracellular and intracellular ionic changes that occur during cerebral ischemia. Upon exposure to K/O, astrocytes swelled slowly and progressively with no evidence of volume regulation. The K/O‐induced swelling was inhibited by 65% with bumetanide and 25% with BaCl2, suggesting contribution of Na+/K+/Cl− co‐transporter and Kir channels. K/O‐elicited 3H‐d‐aspartate that consisted of two phases. The first transient component of the release corresponded to 13.5% of total 3H‐d‐aspartate loaded. It was markedly reduced (61%) by the glutamate transporter blocker DL‐threo‐b‐Benzyloxyaspartic acid and weakly inhibited (21%) by the volume‐sensitive anion channel blocker 4‐[(2‐Butyl‐6,7dichloro‐2‐cyclopentyl‐2,3‐dihidro‐1oxo‐1H‐inden‐5‐yl)oxy] butanoic acid (DCPIB). During the second sustained phase of release, cells lost 45% of loaded of 3H‐d‐aspartate via a mechanism that was insensitive to DL‐threo‐b‐Benzyloxyaspartic acid but nearly completely suppressed by DCPIB. Thrombin (5 U/mL) had only marginal effects on the first phase but strongly potentiated (more than two‐fold) 3H‐d‐aspartate efflux in the second phase. The effect of thrombin effect was proportional to cell swelling and completely suppressed by DCPIB. Overall our data showed that under K/O swelling conditions, thrombin potently enhance glutamate release via volume‐sensitive anion channel. Similar mechanisms may contribute to brain damage in neural pathologies which are associated with cell swelling, glutamate efflux and increased thrombin levels.

Taurine enhances proliferation and promotes neuronal specification of murine and human neural stem/progenitor cells.

Brain development is impaired in taurine deficient animals, showing neuronal delayed maturation and migration. To get insight in the mechanism of this requirement, our studies examined the effect of taurine deficiency in stem/progenitor cells (collectively named neural precursor cells, NPCs). NPCs obtained from mesencephalon of mice embryos (E13.5 days), the subventricular zone of adult mice or human fetal brain, were cultured in media containing EGF and bFGF, and grown as neurospheres. NPCs become taurine-deficient after few days in culture. Addition of taurine replenished the cell pool by a taurine transporter, functionally expressed in NPCs. Taurine-containing cultures contain higher number of cells, due to increased proliferation, evaluated by BrdU incorporation and flow cytometry DNA analysis. Taurine effects are not immediate, requiring a long time interaction with cells. Taurine is not present in cell nuclei, discarding a direct action on nuclear elements. Taurine containing cultures show a higher number of cells with more efficient mitochondrial potential, as detected by flow cytometry assays using rhodamine123/NAO and JC1. A microarray analysis revealed that taurine regulates NPC genes implicated in proliferation, survival, adhesion and mitochondrial functioning. NPCs cultured in medium with bovine fetal serum differentiate into astrocytes, neurons and oligodendrocytes. A markedly high number of neurons were found in taurine cultures from NPCs from adult murine brain (229 %) and human fetal brain (307 %). This effect may also be a consequence of a better mitochondrial functioning as neuronal survival in cultures is markedly affected by the energy state of the cell during the differentiation phase.