Ion Channels and Neuromics’ STEMEZ Cells

In my conversation with neuro-drug discover researchers, I am frequently being asked about the potential of using our STEMEZ(TM) hNP1 Human Neural Progenitors Expansion Kits for studying ion channels. How effective are these cells as a source for studying neurodegenerative diseases and for drug screening assays?  There is good news from Dr. Steve Stice and my friends from ArunA and UGA.

When differentiated, these  neural progenitors express subunits of glutamatergic,  GABAergic, nicotinic, purinergic and transient receptor potential receptors. In addition, sodium  and calcium channel subunits were also expressed. Functionally, virtually all the NP cells exhibited delayed rectifier potassium channel currents and some differentiated cells exhibited  tetrodotoxin sensitive, voltage-dependent sodium channel current under whole-cell voltage clamp and action potentials could be elicited by current injection under whole-cell current clamp.  These results indicate that removing basic fibroblast growth factor from the neural progenitor cell cultures leads to a post-mitotic state, and also results in the capability to produce excitable cells that can generate action potentials. This is the first data demonstrating capabilitiesof these cells for ionotrophic receptor assays and ultimately for electrically active human neural cell assays for drug discovery.
hNP1_Gene_Expression

Images: Glutamate receptor expression in hNP cells and differentiated hNP cells The expression of ionotropic glutamate receptors might also be an indicator of neuronal maturation. These receptors are composed of three distinct families: NMDA, kainate and AMPA receptors. The hNP cells and differentiated hNP cells cultured in the absence of bFGF for 2 weeks were analyzed for mRNA expression of subunits of each glutamate receptor subtype relative to hESCs. Significant increases (p<0.05) in Grin2b were seen in hNP cells (20 fold) and differentiated hNP cells (25 fold) relative to hESCs (Figure 3A). Additionally, Grin1 and Grin2d were significantly increased (p<0.05) only in differentiated hNP cells relative to hESCs, but not in undifferentiated hNP cells (Figure 3A). Of the kainate receptors, Grik4 and Grik5 were significantly (p<0.05) increased only in undifferentiated hNP cells relative to hESCs (Figure 3B); whereas, Grik2 was significantly (p<0.05) increased only in hNP cells where bFGF had been removed (Figure 3B). AMPA receptor subunits were also examined. Gria1 and Gria4 were up regulated in hNP cells relative to hESCs (Figure 3C). Two week differentiated hNP cells showed significant (p<0.05) up regulation of Gria2 and Gira4 relative to hESCs (Figure 3C). To determine if functional glutamate channels exist in differentiated hNP cells, calcium influx in response to AMPA, kainic acid or NMDA application was measured on hNP cells, 14 days after the removal of bFGF. Figure 3G indicates that NMDA could not depolarize differentiated or undifferentiated hNP cells enough to cause significant calcium influx above background. In contrast, AMPA and kainic acid can cause calcium influx which can be potentiated by AMPA receptor specific modulator, cyclothiazide (50 μM, Figure 3G).Calcium influx was detected in the presence of cyclothiazide in calcium activity as measured (Figure 3H).

hNP1_Electrophysiology

Images: Sodium channel activity in differentiated hNP cells was measured using whole cell voltage clamp. 81 total hNP cells cultured in the absence of bFGF from 4 to 27 days were analyzed. Of these, 34 exhibited no fast inward currents in response to a step depolarization indicating the 348 absence of functional voltage gated sodium channels (Figure 4G). The remaining cells yielded between 0.04 – 1.5 nA of inward current in response to the step depolarization (Figures 4B and 4G). These currents inactivated rapidly in all cases (Figures 4B and 4C) and could be abolished with the addition of 1 μM TTX (n = 3 cells; Figure 4C). Voltage-dependent steady state inactivation (n = 11 cells; Figure 4D) and recovery from fast inactivation (n = 5 cells; Figure 4E) were also observed on several positive cells. A subset of these cells was subjected to current clamp and action potentials were elicited by current injection (n = 8 cells, Figure 4F). In support of this, increasing concentrations of a sodium channel activator veratridine in a FLIPR assay on differentiated hNP cells show an increasing calcium response (Figure 4H). This probably resulted from voltage-gated sodium channel depolarization of cells that subsequently allowed calcium influx through calcium channels. These data indicate that differentiation of hNP cells by removal of bFGF can lead to a neuronal cell that can generate action potentials and depolarize the cell. The 58% hit rate for voltage-gated sodium channel function (Figure 4G), does not reflect the true proportion of sodium channel positive cells in our differentiated hNP cells, but rather our ability to morphologically distinguish these cells from negative cells by eye. An example of the morphology of a sodium channel positive cell is shown in Figure 4A. The positive cells were phase bright with a few long processes.

Featuring Dr. Pat Carr

Amyotrophic Lateral Sclerosis (ALS)-New Twists on Root Causes

Teacher, Mentor and Friend    Dr. Pat Carr has been a key figure in helping shape the direction of my company. He has a gift for communicating the nuances of his research and coaching me on how to best serve labs like his. Based on these interactions, it came as no surprise to learn of his being Recognized for Excellence in Teaching, Research and Service at University of North Dakota.

“Dr. Carr has a magic way of teaching,” said second-year medical student, Tyson Bolinske. “He is able to take the most difficult topics and, through detailed notes, logically break down the material.

From a recent dialog, I learned of his growing work on the Ventral Horn and search for root causes of Amyotrophic Lateral Sclerosis (ALS).   I wanted to learn more! I would like to thank Pat for agreeing to share his story and giving me the opportunity to feature highlights in  “News Behind the Neuroscience News”.

 Information on ALS

ALS is an insidious disease.  It is a progressive neurodenerative disease that is always fatal. Approximately 5600 new cases are diagnosed each year. Average survival is typically 3-5 years from onset. The most common form of ALS in the United States is “sporadic” ALS. It can happen to anyone at anytime.  The other is the inherited form named “Familial” ALS (FALS). Only about 5 to 10% of all ALS patients appear to have FALS. As the disease progresses the symptons become more acute. Paralysis spreads through the body affecting  speech, swallowing, chewing and breathing. Ventilator support is need in late stages

 Pat’s Journey

Pat took the “road less traveled”.  He was a passionate hockey player in Canada. He  concluded in his late teens that he was not at a level to take this road to wealth and fame.

Pat Carr

Pat Carr

06/04–present Associate Professor, Department of Anatomy & Cell Biology, School of Medicine and Health Sciences, University of North Dakota 

1996–98 Research Associate/Adjunct Assistant Professor/Auxilliary Assistant Professor, Department of Anatomy;Wright State University

 07/98–06/04 Assistant Professor, Department of Anatomy & Cell Biology, School of Medicine and Health Sciences, University of North Dakota

Postdoc, National Institutes of Health, Neuroscience, 1994-96

Postdoc, University of Manitoba, Neuroscience, 1992-1994    

Ph.D., University of Manitoba, Physiology, 1992

Next was a stint as an automechanic in Brandon, Canada. The discipline and logic involved in fixing cars catalyzed an interest in Science which led to him going to Brandon University to study Geology. When the oil market collapsed in 1983, he decided to change his studies to Zoology and earned a BS in 1984.

A passion was sparked when he did field research in the Canadien Rockies studying parasites in Columbian Ground  Squirrels. He loved it, but recognized the limited value of continuing thsese studies. This lead to the wide open field of Neuroscience and the opportunity to study and solve problems that could benefit mankind. His graduate work at University of Manitoba and focusing on Neuropathic Pain and the Dorsal Horn. He then moved on to studying Ventral Horn and Motor Control Function for his Post Doc at Wright State.

From Pain to ALS

It was Pat’s work in Pain at the University of North Dakota that brought me into initial contact with him. He generously put some of our key Pain/Inflammation and  Neurotransmission Research Antibodies through their paces. These included some of our Neuropeptide and Neuropeptide Receptors , P2X Receptors and TRPV1s (Vanilloids).

His previous work in studying the Ventral Horn combined with a colleagues mouse model of ALS combined to create a prefect opportunity to advance the understanding of ALS.  Pat cautioned me with this insight:  ”sometimes it is  not what you want to study; it is what you can study.  The model is  SOD1 (superoxide dismutase 1) which is core to FALS.(occurs in only about 10% of the ALS cases).

Pat is broadening the play field by looking at what else is happening in sporadic ALS vs FALS. Specifically, he is looking at modulation of alpha Motor Neurons and how the activity of adjacent Renshaw Cells impact signaling and modulation.  Renshaw Cells act as a “governor” on the activity of these alpha Motor Neurons. 

He is drilling down by studying the signaling of ChAT (Choline Acetyltransferase), VAChT (Vesicular acetylcholine transporter) and related molecules. By gaining a deeper understanding of how Renshaw Cells signaling changes the activity of alpha Motor Neurons in ALS,  Pat and his team are taking steps towards discovering roots causes.

As these root causes are further illuminated, I will be reporting specifics in my blog.

The Quest for Better Pain Therapies

G- protein coupled receptor (GPCR) and Drug Responsiveness

About Dr. Laura Bohn 

bohn1

Dr. Laura Bohn

 Background:

Spring 2009-Associate Professor (tenured) at The Scripps Research Institute, Department of Molecular Therapeutics, Jupiter, FL.

10/2007- Associate Professor (tenured), The Ohio State University College of Medicine, Departments of Pharmacology and Psychiatry, Program in Pharmacogenomics

8/2003-9/2007 Assistant Professor, The Ohio State University College of Medicine,

1/1999–8/2003 Post-Doc/Assistant Research Professor. Duke University Medical Center, Department of Cell Biology. Durham, NC. 

 

 

My company’s foundation is built on serving pain researchers. As a result, I have the good fortune of working with customers and collaborators who openly share the subtleties of their research and the future impact it could have on improving pain therapies.

Pain is complex. Today, pain therapies often fall short and are rife with unwelcome side effects. This undesrcores why I am pleased to feature Dr. Laura Bohn. She and her team are probing ways to improve  response effectiveness and reduce side effects.

 Beginnings

The story starts with Laura’s Post Doc work in Dr. Marc Caron’s lab at Duke University.  Marc in Collaboration with Dr. Dr. Robert Lefkowitz genetically engineered mice that lacked a protein switched called  “beta-arrestin 2.”  This switch is part of the opioid pathway that regulates how we perceive pain. The GPCR, muOpioid (mOR) is the primary target for narcotic pain killers, like morphine.

In her initial work, Laura found that morphine treated mice lacking the beta-arrestin2 switch swere able to tolerate  mild pain stimuli up to 3X longer than normal mice.  These mice had a higher level of sensitivity to morphine both in magnitude and duration. 

Bingo. This path for Laura’s excellent journey is now lit…understanding how the molecular regulation of G protein coupled receptors (GPCR) can translate to overall drug responsiveness in vivo.  Getting better response from lower dose is all good.

Current Work

As a researcher at Ohio State University, Laura and her team have continued to broaden and deepen their understanding of  GPCR signaling and beta-arrestin desensitivation (figure 1).

gpcr_regulation 

She is currently doing research with mice that have genetic deletions of GRKs (GRK3, GRK4, GRK5, and GRK6; heterozygotes for GRK2) and barrestin-2.

This expands the playing field. This expansion includes  studying other GCPR related pathways. Serotonin 2A receptors (5-HT2ARs), for example, are molecular targets for drug-induced hallucinations:

Cullen L. Schmid, Kirsten M. Raehal, and Laura M. Bohn. Agonist-directed signaling of the serotonin 2A receptor depends on β-arrestin-2 interactions in vivo. Published online on January 14, 2008, 10.1073/pnas.0708862105.

The conclusion: 5-HT2AR–β-arrestin interaction may be particularly important in receptor function in response to endogenous serotonin levels, which could have major implications in drug development for treating neuropsychiatric disorders such as depression and schizophrenia.

Future Considerations

I look for Laura and her team to continue the quest of doing more for less when it comes to novel pain and other therapies. Further success would provide the foundation for the development of therapies that would require less dosing, better response and reduced side effects.

Laura mentioned to me that further directions could involve the use of gene silencing tools like siRNA. The effects of silencing GPCR-beta-Arrestin receptors in-vivo would be an important study as it would enable she and her team to study  impact of  desensitivation on the repsonse to morphine and other drugs by normal mice.

ACIC3 Receptors Knockdown in vivo

Researchers using siRNA complexed with our i-Fect ™ transfection regent have successfully knocked down ASIC3 Receptors in vivo. This publication joins the growing parade (starting with Luo et al, 2005) that reference successful modulation of receptors involved in pain using siRNA complexes. These studies all share animal behavior studies showing a marked change in response to pain stimuli after treatment.

In this study, Dr. Eric Lingueglia and his team found Peripheral ASIC3 channels are thus essential sensors of acidic pain and integrators of molecular signals produced during inflammation where they contribute to primary hyperalgesia.

Emmanuel Deval, Jacques Noël, Nadège Lay, Abdelkrim Alloui, Sylvie Diochot, Valérie Friend, Martine Jodar, Michel Lazdunski and Eric Lingueglia. ASIC3, a sensor of acidic and primary inflammatory pain. The EMBO Journal advance online publication 16 October 2008; doi: 10.1038/emboj.2008.213

 Cy3-labelled siRNA no. 1121 and its corresponding scramble (no. 1121S; GCTCACACTACGCAGAGAT) synthesized by MWG Biotech (Germany) were injected in rats by intrathecal bolus to the lumbar region of the spinal cord once a day for 3 days before the induction of inflammation with CFA. Each 10-ml injection corresponded to 2 mg of siRNA complexed with i-Fect siRNA transfection reagent (Neuromics) at a ratio of 1:4 (w:v) (Luo et al, 2005), following the supplier’s suggested protocol. siRNA uptake in lumbar DRGs
was monitored by fluorescence microscopy on cryostat sections 24 h after a single intrathecal injection.

Here’s a synopsis of results:

Inflammation was produced by CFA injection, which led to primary heat hyperalgesia, and this hyperalgesia was drastically reduced by the ASIC3 blocker APETx2 injected subcutaneously, which only access cutaneous nociceptors. It was also drastically reduced when, before triggering the inflammation state, intrathecal
injections of an siRNA against ASIC3 had induced a knockdown of ASIC3 expression in lumbar DRGs.

I will continue to publish updates.

Dr. Philippe Sarret Team and Potential New Pain Targets

Shedding Light on New Pain Pathways

There is no joy in Painville. Our answer to pain is: “make it go away”! It spoils quality of life. The socio-economic costs for treatments, loss of productivity and absenteeism, are measured in billions USD$.

Today, moderate to severe pain is treated mostly with NSAIDs, narcotics or tricyclics (anti-depressants). Properly prescribed, these effectively alleviate pain. However, for cases of sustained chronic pain, they become problematic. More than 30% of the population coping with chronic pain are insensitive to morphine derivatives or other pain treatments. They can lose their effectiveness (tolerance), most can be abused and are addictive (dependence), but overall, given in multitherapy, their side effects are additive and deleterious. These problems arise from a lack of comprehension in their mode of action. This is not good news for neuropathic and chronic pain sufferers looking for long term relief.

Research that could lead to discovery of non-narcotic drugs signaling via opioidergic-independent pathways is part of the solution for people coping with chronic pain. This brings us to our back story featuring Dr. Philippe Sarret and his Research Team at the University of Sherbrooke.


About Dr. Philippe Sarret

-Masters (biochemistry), University of Nice in 1994.

-Diploma (DEA, cellular and molecular biology), University of Nice 1996.

-PhD (pharmacology), Institute of Molecular and Cellular Pharmacology, Sophia Antipolis 2000

-Post-doctorate (Neuroscience), Montreal Neurological Institute (MNI), McGill University, Montreal 2004.

-Professor, Faculty of Medicine and Health Sciences, University of Sherbrooke in 2004 -present

Sarret Website-In English

Sarret Website-In French

Tél.: (819) 820-6868, poste 12554
Téléc.: (819) 820-6887
Courriel: Philippe.Sarret@USherbrooke.ca

I asked Dr. Nicolas Beaudet, a Sarret lab member, why he joined the lab. He said, “ Philippe is a great communicator. He has the ability to articulate his complex research in a way that is easy to understand, visionary and exciting”. The aspect that Nicolas finds most intriguing is the systems approach that Philippe and the team take in understanding the mechanisms of pain. This enables them to work at them molecular level up to the whole animal. This is a key step in finding potential new pain therapies.

Drilling Down

Philippe and his team centered their efforts on G Protein Coupled Receptors (GPCRs) such as apelin, chemokines and neurotensin. As a common point, they were all recently identified in the central nervous system to provide a potential role in pain modulation.

Lately, the focus has been on the roles of Neurotensin Receptor 1 (NTS1) and Neurotensin Receptor 2 (NTS2). Recent studies have highlighted the role of these receptors in pain modulation and more is to come…:

  • Geneviève Roussy, Marc-André Dansereau , Louis Doré-Savard, Karine Belleville, Nicolas Beaudet, Elliott Richelson and Philippe Sarret. Spinal NTS1 receptors regulate nociceptive signaling in a rat formalin tonic pain model.Journal of Neurochemistry 105: 1100 – 1114
  • Sarret, P, Perron, A, Stroh, T and Beaudet, A (2003). Immunohistochemical distributionmof NTS2 neurotensin receptors in the rat central nervous system. J Comp Neurol 461: 520–538.
  • Sarret, P, Esdaile, MJ, Perron, A, Martinez, J, Stroh, T and Beaudet, A (2005). Potent spinal analgesia elicited through stimulation of NTS2 neurotensin receptors. J Neurosci 25: 8188–8196.
  • Dobner, PR (2006). Neurotensin and pain modulation. Peptides 27: 2405–2414.
  • Maeno, H, Yamada, K, Santo-Yamada, Y, Aoki, K, Sun, YJ, Sato, E et al. (2004). Comparison of mice deficient in the high- or low-affinity neurotensin receptors, Ntsr1 or Ntsr2, reveals a novel function for Ntsr2 in thermal nociception. Brain Res998: 122–129.      

The wow factor for me was the clever way Philippe and his team used a new technology of 27mer NTS2 Dicer Duplex siRNA (DsiRNA) delivery in vivo as a proof for the potential of DisRNAs-based pain therapies.

Louis Doré-Savard, Geneviève Roussy, Marc-André Dansereau, Michael A Collingwood, Kim A Lennox, Scott D Rose, Nicolas Beaudet, Mark A Behlke and Philippe Sarret. Central Delivery of Dicer-substrate siRNA: A Direct Application for Pain Research. Molecular Therapy (2008); Jul;16(7):1331-9. Epub 2008 Jun 3 doi:10.1038/mt.2008.98.

Using ultra low dose of DsiRNAs complexed with Neuromics’ i-Fect ™, they were able to successfully reduce NTS2 gene expression by up to 86% in rat lumbar Dorsal Root Ganglia after only two intrathecal injections. This was confirmed by Western Blot and qPCR analysis.

What Happened

Using an acute pain model, anti-nociceptive effects of NTS2, induced by a selective agonist, were significantly reduced following NTS2 silencing This resulted in rats showing an increased sensitivity to pain. By day four, the knockdown effects showed a decrease with the NTS2 function returning to normal.

What ‘s next

So we have a great start. We know that agonists binding to NTS2 in the CNS lead to analgesia. We know that DsiRNA can be used to alter the expression of this gene in vivo. We have provided a key step in learning how the NTS2 receptors can be manipulated to block pain. However, now we need to unravel the underlying mechanisms explaining these spinal analgesic properties.

It is my hope that Philippe and his team are appropriately funded. This would catalyze further discoveries in how expression of G Protein Coupled Receptors like NTS1, NTS2, APJ, CCR2 can be targeted to modulate pain. By using rodents, the team can develop tools like DsiRNA to increase the potency and duration of pain blockade. Moreover, potential toxicity and side effects need to be addressed in order to move forward towards clinical studies. These pre-clinical models prove invaluable in taking the step to studies in humans.These therapies hold the promise of providing relief for chronic pain (neuropathic, arthritic, diabetic, cancer pain, etc.) sufferers without the current side effects. Stay tuned as I will be reporting the good news as it unfolds.

Making Gains on Pain

Nicolas Beaudet

Nicolas Beaudet

Neuromics has been helping researchers make “gains on pain” from day one. Our initial sales were Opioid Receptor Antibdies licensed from Dr. Robert Elde’s lab at the University of Minnesota.

From this start, we have expanded our expertise and products. Our reputation in this area has resulted in our forming collaborations and friendships with research teams doing important work in chronic and nociceptive pain research.

Over the past several years, I have worked closely with Dr. Nicolas Beaudet, a member of Dr. Philippe Sarret’s team, at the University of Sherbrooke”. In our next backstory we will feature the vangaurd work they are doing on the Neurotensin (NTS) Receptors and pain.

Manipulation of these receptors-NTS-1, NTS-2 and NTS-3 could represent a therapy for pain independent of the opioid pathway. This means pain could be treated without narcotics meaning a reduction of side effects from current treatments including addiction to pain killers.