Gerry Shaw-Master of World Class Neuronal/Glial Markers

Build it and They will Come

Gerry and One of His Triumph's MCs
Gerry and One of His Triumph’s MCs

I am pleased to profile Dr. Gerry Shaw, a Professor at the University of Florida and also the Head of EnCor Biotechnology Inc.  His story is a guide for incubating and spinning out a successful biotech company (EnCor Biotechnology, Inc.) from a university research laboratory. It should provide an inspiration for fledgling entrepreneurs as the model required little capital investment and has enjoyed profitable growth.

The Backstory

Gerry’s major area of research interest can be summarized as the study of cellular changes resulting from central nervous system damage and disease states. These changes help neuroscience researchers understand the progression and hopefully discover root causes of diseases like Alzheimer’s, Parkinson’s and ALS. Understanding which proteins are involved in particular disease states also has the potential of identifying targets for therapies.

The story starts with Gerry’s Post Doctoral research at the Max Planck Institute for Biophysical Chemistry in Goettingen, in what was at the time West Germany. Here he joined the world renowned laboratory of Klaus Weber and Mary Osborn. This lab had pioneering several important techniques, notably SDS-PAGE for protein analysis and the use of antibodies in immunocytochemistry. Later, after Gerry left the same lab made key contributions leading to the routine use of RNAi in “knock down” of normal cellular proteins. The lab had developed antibodies to tag the subunit proteins of microtubules, microfilaments, intermediate filaments and other cellular proteins, and then used these antibodies to visualize the proteins in immunofluorescence microscopy and on western blots. This enabled researchers to look at changes in the cellular expression of these proteins in powerful new way. These methods have become vital tools for understanding normal cellular function and what happens when cells transition from healthy to diseased states. This lab was an ideal location for Gerry to learn how to make quality monoclonal and polyclonal antibodies. Good antibody reagents are vital for the correct interpretation of immunofluorescence microscopy and western blots, and he was soon supplying his reagents to friends, collaborators and other researchers all around the world. Success is value as antibodies that do not as work as expected waste research time and resources, while quality reagents soon become appreciated and may get to be standard lab reagents.

University of Florida

The University of Florida, in Gainesville imported his expertise when Gerry joined the institute in 1986. Here he continued to make antibodies to Neurofilaments or NFs and other Neuronal-Glial Markers. It’s hard to keep a good thing a secret and Gerry faced growing demand from all over for these reagents. This proved a drain both financially and in terms of time commitment, as well as a significant conflict of interest with his basic biomedical research program.

MAP2_Doering IHC Image: Co-culture of embryonic mouse hippocampal neurons and astrocytes. Primary embryonic hippocampal neurons at 7 days in vitro, were stained with Microtubule Associated Protein-2 (MAP, green) to enable the visualization of the dendritic arbors. These neurons were cultured on top of a monolayer of primary cortical astrocytes, stained with an antibody directed against

Glial Fibrillary Acidic Protein (GFAP, red). The cell nuclei were visualized by staining with 4′,6-diamidino-2-phenylindole (DAPI, blue). BMC Image of the Month October 2010

As a result Gerry took his first entrepreneurial step by selling his most popular reagents in bulk initially to Chemicon (now Millipore-Merck). Like any new business venture, he did not really know what to expect. It should come as no surprise that the reagents sold like hot cakes and the check started rolling in. Other immunoreagent companies approached Gerry and soon he was supplying antibodies to pretty much every major biotechnology vendor.

ABC Biologicals to EnCor Biotechnology Inc.

Success breeds success and as sales increased over the 1990s, it was time to form an independent business and so ABC Biologicals Inc. was incorporated in 1999 initially to buy equipment and develop licensing agreements. Since Gerry had income from sales, he was in the unusual and enviable position of not needing grants, investors, loans or cash from any other source, and so could proceed with almost total independence. The company was renamed EnCor Biotechnology Inc. in 2002, and at the same time moved into the Sid Martin Biotechnology Incubator, a lab dedicated to commercialization of intellectual property generated by the faculty of the University of Florida. The University of Florida is unusually experienced at this and is well known for launching Gatorade, Trusopt and many other products. After 4 years EnCor “graduated” from the Incubator and now occupies a facility in Gainesville. The company now has almost 100 products with many more under development. This is good news for the Neuroscience community.

The EnCor-Neuromics Connection

Neuromics provides EnCor Biotechnology reagents to researchers studying neuro-degeneration, neuro-regeneration, neuro-development, neural stem cells, mood disorders, brain injury and spinal cord injury. My customers have found EnCor’s reagents to be rock solid and versatile.

In addition, Gerry and his team have proved adept at culturing our E18 hippocampal neurons and ESC derived hN2TM primary neurons. This is a big plus as we can actually see how the cells and markers could resonate together for use in cell based assays.

Hippo_MAPT_DC1 Image: E18 hippocampal neurons stained with Tau (red) and Doublecortin (green). The two proteins overlap in the proximal dendrites (yellow) Axons (low doublecortin content) are red. Blue staining is the nuclear DNA.

Futures

I am excited by the glimpse of the future that Gerry shared. We can expect many new, novel and important markers in the coming months and years. In addition, he will be manufacturing various Enzyme-linked immunosorbent assays (ELISA). These kits have the potential to help clinicians diagnose the early onset of diseases like ALS, Parkinson’s and Alzheimer’s.

For example, his company currently sells an ELISA kit for sensitive detection of Phosphorylated Neurofilament-H (pNF-H). Expression of this protein is up regulated in a variety of damage and disease states, and can be used to accurately quantify this up regulation. The kit can also detect pNF-H in the sera and spinal cord fluid (CSF) of animals with spinal cord and brain lesions. This protein is not normally found in sera or CSF, so its presence indicates recent axonal injury as a result of either damage or disease. This suggests pNF-H is a useful biomarker of neuronal and more specifically axonal injury or degeneration, a suggestion supported by a growing list of basic science publications on various animal models and patient types from Gerry’s research lab (e.g. Shaw et al. 2005, Lewis et al. 2008, Boylan et al. 2009, Lewis et al. 2010).

Given the capabilities of EnCor’s markers, the development of more kits is coming. There could be a day in the not distant future where they give clinicians tools to better diagnose and monitor serious neurodegenerative diseases, leading to better disease treatment and management.

I will keep you informed on Gerry’s and EnCor’s future developments.

Consistent Human Neurons

We have featured Dr. Steve Stice here. He and his team at UGA and Aruna Biomedical are developing products that are highly desired by Neuroscience Researchers.

We are in the process of finalizing details for distributing their human neuron cultures. Here is the related press release:

ArunA Biomedical, Inc. announces alliance with Neuromics for distribution of normal human neural cells.

Athens, Georgia – - March 23, 2009 – - ArunA Biomedical, Inc., announced today an agreement with Neuromics, Inc. of Edina, MN, giving Neuromics the right to non-exclusively market and sell the ArunA hN2™ Human Neural Cells and Neural Culture Medium to support applications in neurological research.ArunA has an exclusive worldwide license to develop and commercialize neural cells derived from human embryonic stem cells (hESC), and hN2 is a second generation product from this technology. These cells offer a consistent population of normal human neural cells that the neural research and pharmaceutical market highly desires.

 “ArunA has further developed its adherent monolayer technology by creating hN2™, a normal human neural cell ideal for drug screening, toxicology studies and basic neural research, and we are pleased to have Neuromics as a distribution partner,” said David Ray, Chief Executive Officer  of ArunA Biomedical

“Neuromics growth is catalyzed by offering the unique products and expertise our customers require for research success through strategic alliances with companies like ArunA Biomedical. This relationship represents a growth opportunity for us. Their hN2™ cells fill a stated research need of the Neuroscience Community and we look forward to our customers having these cells and the related new discoveries they will help generate,” said Pete Shuster, CEO and Owner of Neuromics.

Founded in 2003, ArunA Biomedical, Inc. is a privately held biotechnology corporation dedicated to the discovery, manufacturing and commercialization of emerging new technologies in human embryonic stem cell research for use in drug discovery and neuroscience research.

Founded in 2003, Neuromics is a privately held Bio-regents Company focusing on providing research ready and proven products and methods expertise to Neuroscience, Diabetes/Obesity, Immunology and Researchers.
 
This press release contains forward-looking statements regarding the company’s potential impact on scientific research and collaborations with third parties.  Certain conditions could alter the outcome or progress of these statements including but not limited to unexpected manufacturing issues, product performance and quality control/assurance issues.  Forward- looking statements are based on the opinions, beliefs and expectations of the company or individuals quoted in the press release and the company does not assume any obligation to update these forward-looking statements if circumstances change. 

Spinal Cord Injury and Substance-P

I have featured Dr. Matt Ramer  and his research on Spinal Cord Injury. The focus was sensory and autonomic neuron repair.

As an update, I would like to share a publication that references SCI and our Substance-P antibody. Here Dr. Paul Dolber et al. found Substance P detected immunohistochemically in the sacral parasympathetic nucleus was significantly higher in 12 SCI rats than in 12 spinally intact rats (P = 0.008), suggesting substance P as a plausible candidate for the primary afferent neurotransmitter. raises the possibility that substance P may be important in the afferent limb of the spinal micturition reflex that develops about a week after spinal cord transection. This could provide a clue to alleviating urinary disorders related to SCI.

Xiaoyang Zhang, Kristy L.Douglas, Huixia Jin, Bassem M. Eldaif, Rashid Nassar, Matthew O. Fraser, and Paul C. Dolber. Sprouting of substance P-expressing primary afferent central terminals and spinal micturition reflex NK1 receptor dependence after spinal cord injury. Am J Physiol Regul Integr Comp Physiol 295: R2084-R2096, 2008. doi:10.1152/ajpregu.90653.2008.

…guinea pig anti- Substance-P (cat. no. GP14103, Neuromics, Minneapolis, MN) used at 1:1,000…

The Sensory and Autonomic Side of Spinal Cord Injury

About Dr. Matt Ramer

Matt Ramer

Matt Ramer

  • 2001-Present-Associate Professor-University of British Columbia and ICORD
  • Post Doc-King’s College London
  • PhD.-Physiology-Queen’s College Kingston, Ontario

Matt Ramer Website

Awards and Funding

Email: ramer@icord.org

Lab Members: A. Gaudet, J. Inskip, A. Scott, L. Soril

Finding Fixes for Injured Nerves

I first became aware of Matt’s research in early 2005. This was catalyzed when he kindly shared excellent IHC images his lab generated using our BDNF and NT-3 antibodies. I was impressed with him and his team’s data and related publications. I did not understand the context of his work and the potential future impact on people suffering peripheral nerve and spinal cord injury (SCI) and wanted to learn more.

He has generously taken the time to open up my view on spinal cord injury (SCI) and what are the challenges in finding therapies and cures. I had equated success soley with restoring mobility. I knew little of the bigger complexites and problems faced by sufferers of SCI.

More than 300,000 people in the United States and Canada suffer from SCI. The ecomonic cost is in the 10s of billions. One of the horrors of SCI is lost mobility.

People with SCI also suffer from a host of problems related to loss of senory and autonomic functions. Sensory and autonomic nerves in the periperal nervous systems (PNS) connect to the spinal cord dorsally. This is different and separate from those controlling movement and motor function. A little know fact is autonomic dysfunctions represent the primary causes of morbidity and mortality following SCI.

So what are the functions that are most important to SCI patients and how should they be prioritized for basic research and drug discovery? Here are two publications that provide insight:

Kim D. Anderson, Ph.D. Targeting Recovery: Priorities of the Spinal Cord-Injured Population. October 1, 2004, 21(10): 1371-1383. doi:10.1089/neu.2004.21.1371.

J A Inskip, L M Ramer, M S Ramer and A V Krassioukov. Autonomic assessment of animals with spinal cord injury: tools, techniques and translation. Spinal Cord advances online publication 10 June 2008; doi: 10.1038/sc.2008.61

The Funding Gap

If we look through the eyes of those suffering from SCI, we know that there are a mryiad of health issues that are outside the problem of lost mobility. I fear the public including those responsible for funding define cure as “the paralyzed can walk”. This is evidenced by a gap between motor vs. sensory/autonomic research and priorities. This gap needs to be closed. The work of Matt and his colleagues represents progress and needs to be supported with funding growth. This backstory highlights how research could feed the discovery of therapies that would answer the recovery priorities of SCI pateints. They, after all, know best.

The Backstory

The story starts in 2000 and highlights Matt’s research at King’s College London. This research was done in collaboration with Dr. Stephen McMahon and Dr. John Priestly.

They showed that regeneration in damaged rat sensory neurons was possible. Injured dorsal roots, treated with nerve growth factor (NGF), neurotrophin-3 (NT3) and glial-cell-line-derived neurotrophic factor (GDNF), but not brain-derived neurotrophic factor (BDNF), resulted in selective regrowth of damaged axons across the dorsal root entry zone and into the spinal cord. Dorsal horn neurons were found to be synaptically driven by peripheral nerve stimulation in rats treated with NGF, NT3 and GDNF, demonstrating functional reconnection. In behavioural studies, rats treated with NGF and GDNF recovered sensitivity to noxious heat and pressure:

Matt S. Ramer, John V. Priestley & Stephen B. McMahon. Functional regeneration of sensory axons into the adult spinal cord. Nature 403, 312-316 (20 January 2000) | doi:10.1038/35002084.

This is a tight rope act. While there is opportunity for regeneration, there are also inhibitors to nerve growth at work. Regeneration becomes more problematic as a function of time. Of the neurotrophins that promote regeneration, NT-3 appears to best at combating the competing inhibitory effects of proteins like NOGO-A. These inhibitory proteins are suspected to be secreted by astrocytes and microglia:

Matt S. Ramer, Ishwari Duraisingam, John V. Priestley, and Stephen B. McMahon. Two-Tiered Inhibition of Axon Regeneration at the Dorsal Root Entry Zone. The Journal of Neuroscience, April 15, 2001, 21(8):2651-2660.

Images: Axon growth 2 weeks after rhizotomy plus immediate NT-3 treatment. A, In intact animals, CTB-labeled terminals are present in lamina I and III, but absent from lamina II. B, Regenerating axons grow along the pial surface of the cord and in the superficial laminae of the gray matter, avoiding the degenerating cuneate fasciculus. C, Dark-field micrograph of B. Scale bar: B, 100 µm. D, Dark-field parasaggital section from a 2 week rhizotomized and NT-3-treated rat. E, Same section as in D, immunostained for CTB. CTB-labeled axons can be seen on the pial surface (arrowheads) and within the cord. Many axons have turned to grow in a rostrocaudal direction but appear to do so in the superficial laminae of the gray matter rather than the white matter. Some individual axons can be traced for up to 2 mm. F, In zones in which the density of regenerated axons is greatest, they form a longitudinal bundle in the gray matter, with few axons in the more superficial white matter (arrows). G, Many axons possess terminal swellings that may be growth cones or termination bulbs. Scale bar: E, 300 µm. The Journal of Neuroscience, April 15, 2001, 21(8):2651-2660.

Through the Looking Glass

Matt and his colleagues continue to gain understanding and refine methods for nerve regeneration. They are also studying plasticity and how these neurons connect to sensory and autonomic neurons in the PNS. This is analogous to re-wiring what was once severed. This would enable restoring of functions important to sufferers of SCI. The related good news is that even partial reconnection enable restoration of these lost functions.

Stepping through the looking glass involves understanding the specific role of these neurons. His recent works include:

Matt Ramer. Anatomical and functional characterization of neuropil in the gracile fasciculus. The Journal of Comparative Neurology. 10.1002/cne.21785.

  • Neurokinin-1 (NK 1) Receptor-Detects a band at 80-90 kDa on Western blots of membranes prepared from cells transfected with the rat substance P receptor (Vigna et al., 1994); stainingin rat spinal cord was blocked by preabsorbing the antiserum with the immunizing peptide (Mantyh et al., 1995)-Dilution 1:2,000
  • Substance P-The distribution of immunoreactivity in rat spinal cord is identical to that described previously (Hunt et al.,1981); in dual-labeling experiments, it labels the same structures as a polyclonal rabbit anti-SP (1:1,000; Peninsula/Bachem; T-4107; data not shown).

Here Matt and his team report on the morphology, inputs, projections, and functional properties of these neurons. Small fusiform and larger lentiform neurons are most abundant in the gracile fasciculus of the cervical and lumbar enlargements and are absent from the cuneate fasciculus and corticospinal tract. Many have dendrites that run along the dorsal pia, and, although in transverse sections these neurons appear isolated from the gray matter, they are also connected to area X by varicose and sometimes loosely fasciculated dendrites. These neurons receive neurochemically diverse, compartmentalized synaptic inputs (primary afferent, intrinsic and descending), half express the substance P receptor, and some project supraspinally. Unlike substantia gelatinosa neurons, they do not express protein kinase C gamma. Functionally, they have small receptive fields, which are somatotopically appropriate with respect to their anterior-posterior position along the neuraxis. They respond to innocuous and/or noxious mechanical stimulation of the distal extremities, and some are prone to central sensitization or windup. Morphologically, neurochemically, and functionally, therefore, these cells most closely resemble neurons in laminae III-VI in the dorsal horn.

Closing Thoughts

There is hope for SCI patients. It is clear that related research and funding needs to expand dramatically beyond the current narrow focus on restored motor function and mobility. The priorities are documented and understood. The story continues. Real progress will be marked by answering these priorities with restored function. Sensing pain, pressure, temperature, etc. where today there is only nothingness. Controlling autonomic functions that pose such a risk to SCI sufferers. I will continue to report the progress of Dr. Matt Ramer and his colleagues. Godspeed to them.

Dr. Matthew Ramer, Neural Regeneration and SCI

Dr. Matthew Ramer

Dr. Matthew Ramer

I dream of the day that people suffering from spinal cord injuries (SCIs) will be restored to full function. The good news is there are researchers and dedicated centers that form the back-bone of SCI repair research.

I am excited and honored to be featuring Dr. Matthew Ramer for this month’s backstory. Matt is a member of ICORD (International Collaboration On Repair Discoveries) at the University of British Columbia. ICORD is an interdisciplinary research centre for the development of effective strategies to promote functional recovery after spinal cord injury. This includes  the discovery and implementation of relevant solutions to improve functional recovery, mobility, community integration and quality of life for people with spinal cord injury.

Matt’s research focus is on the molecular biology of primary sensory nerve cells (neurons). These neurons are responible for sensation. These include touch, pain, temperature, etc. These neurons transmit sensation to the brain via the spinal cord. It is this transmission that enables us to process the sensation. Similar transmissions happen in the case of locomotion.

Matt’s research helps us better understand the mechanism of these transmissions. More importantly, his work includes finding ways to regenerate and repair neurons. These are steps in improving the outcome of sufferes of SCI.

Spinal Cord Injury and Ependymal Cells

This research sheds light on the natural regeneration that occurs in the area of damaged spinal cord tissue. The surprise here is the role that Edendymal Progenitor Cells are playing in repair mechanism vs Neural Stem Progenitors. 

In this publication, researchers have employed genetic fate mapping to characterize a candidate neural stem cell population in the adult spinal cord and show that close to all in vitro neural stem cell potential resides within the population of ependymal cells. Ependymal cells give rise to a substantial proportion of scar-forming astrocytes as well as to some myelinating oligodendrocytes after spinal cord injury. Modulating the fate of ependymal cell progeny after injury could potentially promote the generation of cell types that may facilitate recovery after spinal cord injury.

Meletis K, Barnabé-Heider F, Carlén M, Evergren E, Tomilin N, et al. (2008) Spinal Cord Injury Reveals Multilineage Differentiation of Ependymal Cells . PLoS Biol 6(7): e182 doi:10.1371/journal.pbio.0060182