Bidirectional integrative regulation of Cav1.2 calcium channel by microRNA miR-103: role in pain

I have reported use of our i-FectTM siRNA delivery kit for gene expression analysis studies of DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8, , TRPV1, Survivin, Flaviviruses and more.

I am pleased to add the Cav1.2 calcium channel to this growing list. Congratulations to Dr. Marc Landry for discovering the interplay between microRNA-miR-103 and this calcium channel: Alexandre Favereaux, Olivier Thoumine, Rabia Bouali-Benazzouz, Virginie Roques, Marie-Amélie Papon, Sherine Abdel Salam, Guillaume Drutel, Claire Léger, André Calas, Frédéric Nagy and Marc Landry. Bidirectional integrative regulation of Cav1.2 calcium channel by microRNA miR-103: role in pain. The EMBO Journal , (29 July 2011) | doi:10.1038/emboj.2011.249.

Abstract: Chronic pain states are characterized by long-term sensitization of spinal cord neurons that relay nociceptive information to the brain. Among the mechanisms involved, up-regulation of Cav1.2-comprising L-type calcium channel (Cav1.2-LTC) in spinal dorsal horn have a crucial role in chronic neuropathic pain. Here, we address a mechanism of translational regulation of this calcium channel. Translational regulation by microRNAs is a key factor in the expression and function of eukaryotic genomes. Because perfect matching to target sequence is not required for inhibition, theoretically, microRNAs could regulate simultaneously multiple mRNAs. We show here that a single microRNA, miR-103, simultaneously regulates the expression of the three subunits forming Cav1.2-LTC in a novel integrative regulation. This regulation is bidirectional since knocking-down or over-expressing miR-103, respectively, up- or down-regulate the level of Cav1.2-LTC translation. Functionally, we show that miR-103 knockdown in naive rats results in hypersensitivity to pain. Moreover, we demonstrate that miR-103 is down-regulated in neuropathic animals and that miR-103 intrathecal applications successfully relieve pain, identifying miR-103 as a novel possible therapeutic target in neuropathic chronic pain.

MicroRNAs as targets for pain therapies are gathering momentum. This defned miR-103 is a compelling possibilty. I will be following the story closely as it unfolds.

PEI transfection and Implications for a Mechanism of Cytotoxicity

This is a great article for undertanding the root causes of cytotoxicity caused by PEI-DNA Polyplexes.

Giovanna Grandinetti, Nilesh P. Ingle, and Theresa M. Reineke. Interaction of Poly(ethylenimine)–DNA Polyplexes with Mitochondria: Implications for a Mechanism of Cytotoxicity. Mol. Pharmaceutics, Article ASAP. Publication Date (Web): June 23, 2011. Copyright © 2011 American Chemical Society.

Poly(ethylenimine) (PEI) and PEI-based systems have been widely studied for use as nucleic acid delivery vehicles. However, many of these vehicles display high cytotoxicity, rendering them unfit for therapeutic use. By exploring the mechanisms that cause cytotoxicity, and through understanding structure–function relationships between polymers and intracellular interactions, nucleic acid delivery vehicles with precise intracellular properties can be tailored for specific function. Previous research has shown that PEI is able to depolarize mitochondria, but the exact mechanism as to how depolarization is induced remains elusive and therefore is the focus of the current study. Potential mechanisms for mitochondrial depolarization include direct mitochondrial membrane permeabilization by PEI or PEI polyplexes, activation of the mitochondrial permeability transition pore, and interference with mitochondrial membrane proton pumps, specifically Complex I of the electron transport chain and F0F1-ATPase. Herein, confocal microscopy and live cell imaging showed that PEI polyplexes do colocalize to some degree with mitochondria early in transfection, and the degree of colocalization increases over time. Cyclosporin a was used to prevent activation of the mitochondrial membrane permeability transition pore, and it was found that early in transfection cyclosporin a was unable to prevent the loss of mitochondrial membrane potential. Further studies done using rotenone and oligomycin to inhibit Complex I of the electron transport chain and F0F1-ATPase, respectively, indicate that both of these mitochondrial proton pumps are functioning during PEI transfection. Overall, we conclude that direct interaction between polyplexes and mitochondria may be the reason why mitochondrial function is impaired during PEI transfection.

Neuroscience-Best Transfection Practices

I have multiple posting of customer success in transfecting siRNA and plasmids into neurons using Neuromics' i-Fect^TM and pn-Fect^TM...related publications.

Here I post excellent data, images and testimonials provided by researchers using our Magnetic Assisted Transfection (MATraTM). Our goal is to continue our journey towards having the best practices for gene expression analysis studies in the CNS.

With Magnet Assisted Transfection,  IBA/Neuromics offers a very gentle and potent tool for the transfection of many kinds of neuronal cells. Magnet Assisted Transfection is the ideal solution to overcome problems related to the study of complex and easily interrupted systems. Transfection of primary cortical neurons Example 1  Embryonic cortical neurons were transfected with human NCAM. Embryonic cortical neurons were transfected with human NCAM. After transfection membrane-localized NCAM (not endocytosed) was detected using a Cy3-coupled secondary antibody (red). Afterwards, the internalised, endocytosed NCAM was stained by a Cy2-coupled secondary antibody (green, see arrows) in the cell soma (left) and in axonal vesicles (right). Example 2 pPrimary cortical neurons from mice embryonic day 15.5 (E15.5) were grown on poly-L-lysine coated coverslips at a density of 800.000 cells/well in a 24-well plate. The neurons were transfected after 1 day in vitro (DIV 1) with pCX-EGFP-N1 plasmid Primary cortical neurons from mice embryonic day 15.5 (E15.5) were grown on poly-L-lysine coated coverslips at a density of 800.000 cells/well in a 24-well plate. The neurons were transfected after 1 day in vitro (DIV 1) with pCX-EGFP-N1 plasmid. Transfection was carried out as recommended by the manufacturer (0.6 µg DNA, 0.6 µL Matra-A reagent). Cells were fixed 24 h later (DIV 2) and GFP fluorescence was visualized using a confocal laser scanning microscope. "With MATra we achieved a higher transfection efficiency than with different liposomal transfection methods and no toxicity to the cells was observed." Dr. Simone Diestel, Institute of Animal Science, University Bonn, Germany Cerebellar granular cells from CD1 mice  Cultured cerebellar granular cells from CD1 mice were transfected by below 4 constructs Cultured cerebellar granular cells from CD1 mice were transfected by below 4 constructs (A-D) using MATra-A. (A) MyrPalm-mCFP, cyan (provided by Dr. R. Tsien, UCLA) (B) Actin-DsRed, red (C) Flotillin-2-mVenus, yellow (B and C provided by Dr. R. Tikkanen, University of Giessen) (D) Battenin-myc, detected by using GAM-Alexa647, dark green (E) Surface: Crop of the whole image with 3D surface rendered fluorescence signals overlayed on phase contrast image. (F) PhaCo: Phase contrast image Primary hippocampal neurons (E14)

 Primary hippocampal neurons (E14) were grown on 15 mm glass coverslips on a 12 well at density of 150.000/cm². The neurons were transfected 4 d.i.v. with pSyn-eGFP  Primary hippocampal neurons (E14) were grown on 15 mm glass coverslips on a 12 well at density of 150.000/cm². The neurons were transfected 4 d.i.v. with pSyn-eGFP using 25 µl MATra complex per well (prepared by adding a MATra-A Reagent-DNA complex mixture (2.8 µg cDNA; 2.8 µl beads) into 175 µl neuronal medium without serum). The cells were fixed 6 d.i.v. with 4% PFA and imaged "With MATra we can transfect and modulate the expression levels of exogenous proteins in highly sensitive primary neurons without any toxicity. Once optimized, double and even triple transfections with different DNA ratios are easily achieved", said Dr. Mika Ruonala, Center for Membrane Proteomics, University of Frankfurt. Neurosciences are a vast and expanding field of research focussing on highly sophisticated and enthralling questions. With Magnet Assisted Transfection IBA/Neuromics offers a very gentle and potent tool for the transfection of many kinds of neuronal cells. Magnet Assisted Transfection is the ideal solution to overcome problems related to the study of complex and easily interrupted systems.

PKA+siRNA Block Hyperalgesia

I have reported use of our i-Fect^TM siRNA delivery kit for gene expression analysis studies of DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8, , TRPV1, Survivin, Flaviviruses and more.

The data in this pub indicates that selective knock-down of spinal PKA activity by intrathecal (i.th.) pretreatment of rats with a PKA-selective small interference RNA (siRNA) mixture significantly attenuates sustained morphine-mediated augmentation of spinal CGRP immunoreactivity, thermal hyperalgesia, mechanical allodynia and antinociceptive tolerance. The present findings indicate that sustained morphine-mediated activation of spinal cAMP/PKA-dependent signaling may play an important role in opioid induced hyperalgesia: S. Tumati, W.R. Roeskea, T.M. Largent-Milnesa, T.W. Vanderaha, and EV Varga. Intrathecal PKA-selective siRNA treatment blocks sustained morphine-mediated pain sensitization and antinociceptive tolerance in rats. doi:10.1016/j.jneumeth.2011.04.036.

Figure: Intrathecal PKA-selective siRNA treatment blocks the development of morphine antinociceptive tolerance.

Male Sprague Dawley rats were pretreated i.th. with vehicle (inverted triangle) or PKA-selective siRNA (circle) for 3 days. After the pretreatments, the animals received continuous saline (open symbols, error bars within the symbol) or morphine (45 nmol/μl/h) (closed symbols, error bars within the symbol) infusion for 6 days, with continued i.th. siRNA or vehicle injections on alternate days. Sustained (6 days) systemic morphine (45 nmol/μl/h) infusion caused a rightward shift in the dose-response curve, with the previous A90 dose causing only 20±1% MPE (**p < 0.01 relative to control, one-way ANOVA, n=5). Intrathecal PKAselective siRNA pre-treatment greatly attenuated sustained morphine-mediated rightward shift in the morphine dose-response curve. Thus, re-challenge with the naive A90 dose (10 μg/5μl) produced 93±2% antinociception in the PKA-selective siRNA pre-treated rat**p < 0.01 relative to vehicle pre-treated morphine-infused rats, one-way ANOVA, n=5). 

Transfection Kits and Related Reagents:

i-Fect ™ -A novel cationic  lipid formulation specifically designed for efficient delivery of 27mer DsiRNAs(dicer substrate small Interfering RNAs)& 21mer siRNAs (small interfering RNAs) in vitro and in vivo.
n-Fect™ -A cationic lipid that has been specifically formulated for nervous system  applications. n-Fect provides higher transfection efficiency than  other commercially available broad-spectrum transfection reagents for glial cells, neuronal cell lines, and certain primary neuronal  cultures.
n-Blast™ -A broad-spectrum  transfection reagent successfully used in many cell types commonly used by neuroscientist.
 pn-Fect™ -The latest advance in transfection technology for primary neuronal  cells. This unique reagent provides ultra-high plasmid DNA delivery efficiencies and low cytotoxicity compared to competitive reagents.
p-Fect™ -Designed to delivery plasmids, DNA or RNA to hard to transfect Cell Lines.
pro-Fect™ -Is a unique  lipid-based formulation that allows the delivery of proteins,  peptides or other bioactive molecules into a broad range of cell  types.
Penatratin-1™ -A peptide for  delivering small molecules into Neurons and other cells. MP  Biomedical is the manufacturer of Penetratin-1
MATra™ Products-Provides a system for Magnetically Driving the transfection process enhancing
the performance of transfectants.
Other Cells-Competent mammalian cells by Category
Primary Neurons and Astrocytes 

I'll be posting more soon.

ASICs and Surgical Pain

Dr. Eric Lingueglia, an INSERM group leader, and his team at the CNRS IPMC/IN2M have been doing impressive research using our i-Fect ™ siRNA Transfection Kits to study the role of Acid-Sensing Ion Channels in Postoperative Pain.

The etiology and pathophysiology of this pain is poorly understood. Their work is shedding light on potential root causes:
Emmanuel Deval, Jacques Noël, Xavier Gasull1, Anne Delaunay, Abdelkrim Alloui, Valérie Friend, Alain Eschalier, Michel Lazdunski, and Eric Lingueglia. Acid-Sensing Ion Channels in Postoperative Pain. The Journal of Neuroscience, 20 April 2011, 31(16): 6059-6066; doi: 10.1523/​JNEUROSCI.5266-10.2011.

...Ten microliters of a siRNA (2 μg)/i-Fect (Neuromics) mix was injected intrathecally between the L4 and L5 vertebrae of rats using a Hamilton syringe and a 25 gauge needle. Animals received one injection per day for 4 d (Fig. 4A, protocol). ASIC3 (CUACACGCUAUGCCAAGGAdtdt) and the corresponding scramble (GCUCACACUACGCAGAGAUdtdt) siRNAs have been previously described (Deval et al., 2008)...


Highlights: Pharmacological inhibition of ASIC3 channels with the specific toxin APETx2 or in vivo knockdown of ASIC3 subunit by small interfering RNA led to a significant reduction of postoperative spontaneous, thermal, and postural pain behaviors (spontaneous flinching, heat hyperalgesia, and weight bearing). ASIC3 appears to have an important role in deep tissue but also affects prolonged pain evoked by skin incision alone.

ASIC3s are excitatory ion channels directly activated by extracellular protons that detect the painful drops in pH at incision points. Several factors may participate in the drop of extracellular pH, such as release of the acidic content of lyzed cells, degranulation of mast cells, organic acids released by metabolism..etc  This makes makes the Ion Channel a great marker for the studying activation of pain and a potential therapeutic target for mitigating surgical pain.

I will continue to track and report progress.

Delivering TRPV1 shRNA to DRG of T8-L3 Segments of the Spinal Cord

I have reported use of our i-FectTM siRNA delivery kit for gene expression analysis studies of DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8, Survivin, Flaviviruses and more.

This is the first publication referencing the use of i-Fect to delivery shRNA intrathecally. In this study, researchers knockdown TRPV1 Channels in DRGs to study their role in regulation of blood pressure.

Shuang-Quan Yu, Donna H. Wang. Intrathecal injection of TRPV1 shRNA leads to increases in blood pressure in rats. DOI: 10.1111/j.1748-1716.2011.02285.x. Copyright © 2011 Scandinavian Physiological Society.

Aim: The transient receptor potential vanilloid type 1 (TRPV1) channels have been implicated to play a role in blood pressure regulation. However, contribution of tissue specific TRPV1 to blood pressure regulation is largely unknown. Here we test the hypothesis that TRPV1 expressed in dorsal root ganglia (DRG) of lower thoracic and upper lumbar segments (T8-L3) of the spinal cord and their central and peripheral terminals constitutes a counter regulatory mechanism preventing the increases in blood pressure.

Methods: TRPV1 was knocked down by intrathecal injection of TRPV1 shRNA in rats. Systolic blood pressure and mean arterial pressure (MAP) were recorded. The level of TRPV1 and tyrosine hydroxylase was measured by Western blot.

Results: Intrathecal injection of TRPV1 shRNA (6 μg kg−1 per day) for 3 days increased systolic blood pressure and MAP when compared to rats that received control shRNA (control shRNA: 112±2 vs TRPV1 shRNA: 123±2 mmHg). TRPV1 expression was suppressed in T8-L3 segments of dorsal horn and DRG as well as mesenteric arteries of rats given TRPV1 shRNA. Contents of tyrosine hydroxylase, a marker of sympathetic nerves, were increased in mesenteric arteries of rats treated with TRPV1 shRNA. Pretreatment with the 1-adrenoceptor blocker, prazosin (1 mg/kg/day, p.o.), abolished the TRPV1 shRNA-induced pressor effects.

Conclusion: Our data show that selective knockdown of TRPV1 expressed in DRG of T8-L3 segments of the spinal cord and their central and peripheral terminals increases blood pressure, suggesting that neuronal TRPV1 in these segments possesses a tonic anti-hypertensive effect possibly via suppression of the sympathetic nerve activity.

Transfecting Primary Cortical Neurons with a Plasmid for NCAM

Harnessing the power of MATra^TM (Magnetic Assisted) Transfection Kits.

Background: The neural cell adhesion molecule (NCAM) plays a major role during development of the nervous system and in synapse plasticity in the adult brain (Diestel et al., 2007). Many studies provide evidence that NCAM can regulate processes like cell migration, axon growth and fasciculation. Endocytosis of NCAM might play a decisive role in these processes as it can potentially enable a quick change in cell adhesion between the cells or towards the extracellular matrix. Endocytosis of
NCAM might also influence these processes by activating specific signal transduction pathways.

Primary cortical neurons present a good in vitro system for these investigations since they allow analysis of molecules within growth cones. For analysis of NCAM, embryonic cortical neurons (E15.5) were transfected with human NCAM one day after isolation. Endocytosis of NCAM was induced 24 hours after transfection and detected by immunofluorescence analysis.

Results:

Images: Endocytosis of NCAM in cortical neurons. After transfection membrane-localized NCAM (not endocytosed) was detected using a Cy3-coupled secondary antibody (red). Afterwards, the internalised, endocytosed NCAM was stained by a Cy2-coupled secondary antibody (green, see arrows) in the cell soma (left) and in axonal vesicles (right).

The data presented here were provided by Simone Diestel, Institute for Animal Sciences, University
of Bonn, Germany. Published also in "Renker, B. et al. MATra - ein Trojanisches Pferd für eine zellschonende Transfektion. BIOSpektrum 04.10:441-442."
Literature: Diestel S, Schäfer D, Cremer H, Schmitz B. (2007) NCAM is ubiquitylated, endocytosed and
recycled in neurons. J Cell Sci. 120: 4035-49

Material and Methods: Primary cortical neurons from C57BL/6 mice embryonic day 15.5 (E15.5) were isolated and plated at a density of 800,000 cells per 24-well plate on poly-L-lysine-coated coverslips. The next day the neurons were transfected with an expression plasmid for human NCAM by Magnet Assisted
Transfection. To induce endocytosis, 24 hours after transfection cells were incubated 30 minutes at
37°C with an antibody which is specific for human NCAM. Subsequently the cells were fixed and
membrane-localized NCAM was visualized using a Cy3-coupled secondary antibody. After permeabilization of the cells internalised NCAM was stained by a Cy2-coupled secondary antibody. The cells were mounted on microscope slides and analysed using a Zeiss LSM510 MetaUV confocal microscope.

Magnet Assisted Transfection (MATra-A reagent): 0.6 μg DNA were dissolved in 50 μl Neurobasal medium. 0.6 μl MATra-A reagent were added, mixed well and incubated for 20 minutes at room temperature. During this incubation time the  medium was exchanged with supplemented Neurobasal medium (containing B27 supplement and 2 mM L-glutamine). The transfection mixture was added drop by drop to the cells, dispersed evenly in the medium and immediately placed on the magnetic plate (37°C, 5% CO2, 15 minutes). After 6 hours half of the medium was exchanged with fresh, supplemented Neurobasal medium.