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Molecular "Stop Signs" May Hold Secret Of Nerve Regeneration


Johns Hopkins Medical Institutions


Four thousand years ago, the ancient physicians wrote this harsh verdict, we are still unable to change:  spinal cord injury is untreatable. In fact, unfortunately, it's much the same today. Ronald L. Schnaar, Ph.D., professor of pharmacology and of neuroscience at the Johns Hopkins University School of Medicine, says: "But the basic-science framework for improving this situation is quickly emerging."

Using brain cells from rats, neuroscientists at Hopkins and the University of Hamburg have manipulated a molecular stop sign so that the injured nerve cells regenerate. While their findings are far from application in people, the prospects for eventually being able to repair spinal cord injury are brighter, they say.

In adult mammals, including humans, molecular signals carefully control the number of contacts nerve cells make by inhibiting new connections. We know that in the central nervous system, once an axon is interrupted in some way, through disease or injury, generally it's stopped dead in its tracks, but in the rest of the body, damaged axons can re-grow. When the brain or spinal cord has been damaged, the goal is to neutralize those inhibitors so that the axons might re-establish their broken connections. Schnaar says: "To make headway in treating brain and spinal cord injury, we need to attack this problem from a number of angles, and our studies have provided an additional target for intervention."

The research team reports identifying brain chemicals that are involved in the ability of one of the inhibitors to prevent injured nerve cells from connecting to other nerves or muscles. By keeping the chemicals from interacting with the inhibitor, the researchers were able to stimulate damaged nerve cells to regenerate in laboratory dishes. Their report is in the June 11, 2002, issue of the Proceedings of the National Academy of Sciences. 

Of the "stop signs" identified so far, Schnaar's team focused on MAG, or myelin-associated glycoprotein, which is part of the myelin wrapping that insulates all nerve cells. Understanding how the newly identified molecules responsible for MAG's inhibitory effect - gangliosides - interact with MAG to send the "stop" signal to the nerve may lead one day to potential treatments, say the scientists.

In their experiments with rat and mouse cells, Hopkins postdoctoral researcher Alka Vyas tested four ways of stopping MAG and the gangliosides from interacting: destroying part of the ganglioside where MAG usually attaches, limiting the amount of the gangliosides made by the cells, using antibodies to block MAG or using antibodies to block the gangliosides. The research team now is focused on determining exactly how the gangliosides and MAG work together to stop nerve regeneration.


The work was funded by the National Institutes of Health, the National Multiple Sclerosis Society and the Stoll of Family Fund. Other authors on the report are Himatkumar Patel, Susan Fromholt, Marija Heffer-Lauc, Kavita Vyas and Jiyoung Dang of Johns Hopkins; Melitta Schachner of the University of Hamburg, Germany.





Brain Mind & Life




We agree that spinal cord injury (as well as for the entire Central Nervous System) is still untreatable (except for some partial surgical successes in cell transplantation), but we find very hard to agree with the statement: “the framework of the basic science is quickly emerging”.  We rather would say: “A first sketch of the main processes is slowly emerging”. For that, let’s give a glance at the general framework.


In 1928, Santiago Ramon y Cajal reported that Tello showed in 1911 as the Central nervous System perennial neurons could re-grow if they were provided access to the permissive environment of a conditioned sciatic nerve. It took not less than seventy years to come to a decisive confirmation. Aguayo, Richardson and Mc Guinness[1] replicated Tello’s studies with new methods revealing that the failure of central differentiated neurons to regenerate was not an intrinsic deficit of the cell, but rather a characteristic feature of the damaged environment that either did not support or prevented regeneration. In the past twenty years of studies, progress has been made in identifying the elements that are responsible for the differences between the adult Central Nervous System (CNS) and Peripheral Nervous System (PNS) environments, thus in the last decade[2] the molecular and cellular bases of regenerative compared with non-regenerative responses are beginning to be revealed.


The emerging picture of these responses is very complicated and research is divided into several fields of investigation reflecting the different nature of the processes involved. In fact when a chemo-toxic or traumatic event occurs we have alterations on a systemic level, tissue level, cellular level and molecular level. On a cellular level, these deficits include demyelination, degeneration, abortive or aberrant sprouting and cell death.[3]


Demyelination. It always causes trouble in action potential transmission, thus a demyelinated axon may maintain both its afferent and efferent connections but, due to a loss of myelination, poor or failed conduction results.


Axonal Retraction. Damage to the original neuronal target of the axon or to an axon itself can result in degeneration. Pre-synaptic, retrograde (from axon to cell body) and trans-synaptic degeneration can occur. Synaptic conduction across a pathway is lost and a reactive cellular response, including astrocytes and microglia, forms.


Sprouting. Surviving neurons may have a normal axonal sprouting, an aberrant or abortive one. Aberrant sprouting can occur when an axon reconnects to an inappropriate target. Synaptic conduction is restored but this pathway does not result in functional restoration. Abortive, when a sprouting axon encounters an inhibitory matrix or scar, the presence of continuing inflammation or toxicity or the loss of neurotrophin support.


Cell Necrosis. Many different reasons can determine neuronal necrosis, generally as a neuron is completely deprived of its source of growth factors and exposed to high levels of toxic molecules or inflammatory attack, it can undergo cell death.


 These patterns represent the anatomical correlates of brain dysfunction but also the specific processes that must be targeted for repair. Strategies for regenerating the adult central neurons may be described as a multi-step process:


1)      surviving of the injured cell,

2)      axon growth: redirection of axon cut processes to their original neuronal targets;

3)      re-myelination: the axons need to be remyelinated by central glial cells;

            4)   synapse formation: appropriate reconstitution of functioning synaptic connections.


To target different aspect of this multi-step process, there are several strategies as well as different cultural approaches, from cell transplantation to immunology:



1)    Cellular replacement

2)    Neurotrophic factor delivery

3)    Axon guidance and removal of growth inhibition

4)    Manipulation of intracellular signalling

5)    Bridging and artificial substrates

6)    Modulation of the immune response



The manipulation of signalling, at the point “4” of the list, is the field which the study conducted by Schnaar belongs, and it is a broad field. The study is interesting and promising, but we really cannot say that knowing in deep the mechanism of MAG-gangliosides interaction, it will be the decisive turning point in this long adventure. Too many questions have to be answered yet.

 For instance, problems and perspectives derived from the role of the central immune response. For many years there has been speculation about dualism of the immune response to injury in the brain: protective and negative in many ways. On the negative side we have learned that early inhibition of tumour necrosis factors or transforming growth factor-b2 significantly decreases scarring and tissue loss, and can lead to improvement in functional outcome after CNS injury. Application of interferon-g modulates several extracellular matrix molecules found in the CNS scar and inhibits astrocyte proliferation. These observations demonstrate that certain aspects of the immune response may substantially restrict axonal plasticity following trauma.

 Many papers have shown that the presence of neural stem cells resident within the CNS and the ability to regulate their number and fate may provide an alternative to transplantation and molecular focused studies. We know that adult brain and spinal cord contain stem cells that continually divide throughout adult life. With respect to injury, progenitor cells are capable of proliferation and differentiation into mature myelinating oligodendrocytes in models of acute demyelination. In addition, cortical neurons are replaced by dividing progenitor cells in a model of selective pyramidal cell apoptosis. Strikingly, these newborn neurons extend new axons several millimetres through the intact CNS. These findings show that under the correct conditions, CNS stem cells are capable of participating in cell replacement. Despite their inherent plasticity, however, it is clear that endogenous stem cells do not produce complete recovery in cases of severe trauma.

But even this field needs to interact with the others and participate the unsolved questions. In fact whether a strategy of transplantation or endogenous replacement is used, the molecules and genes that govern cell proliferation, migration and differentiation need to be determined. In addition new cells that are grafted or generated in situ will need to be engineered or pharmacologically protected to be resistant to the toxic environment of the CNS under damage effect.

Finally, even when dead cells can be replaced with new ones, the new cells will need to be functionally integrated into the remaining circuitry, by surviving cells and/or by externally driven programs. We need to know those programs.


Among open questions the ones coming from neurotrophic factors are very challenging. Neurotrophins comprise a large and complex family of molecules, and many subclasses of neurons and glia express unique complements of neurotrophic factor receptors. Many questions remain regarding practical application of these molecules; for example, diffusion of exogenously delivered neurotrophins is variable. In addition, neurotrophins have many properties aside from their roles in neuronal survival and axonal growth. For example, neurotrophins are anterogradely transported and released from presynaptic to postsynaptic targets. Neurotrophins also modulate membrane excitability, induce neuronal hypertrophy, affect cell differentiation, and result in broad systemic effects. These functions for neurotrophins will need to be considered when interpreting regenerating studies.

These considerations are largely insufficient for depicting a picture of on going studies, but can help to understand as a cure for spinal cord injury is still out of sight and that it is illusive to think that one molecular mechanism could be the key to solve the entire problem of neural regeneration.  

Horner and Gage review, we broadly referred in our discussion, ends quoting Cajal: “Everything may die, nothing may be regenerated. It is for the science of the future to change, if possible, this harsh decree”. The decree is lifted, but we are just moving the first step in the future[4].



                                                                                                                    Giuseppe Perrella - BM&L

[1] Richardson, P. M., Mc Guinness, U. M. & Aguayo, A. J., Axons from CNS regenerate into PNS grafts. Nature 284, 264-265 (1980).

[2] As early as 1991 a Fred Gage’s work showed the effect of high growth factor levels on reactive astrocyte for the growth of axons: Kawaja M.D., Gage F.H., Reactive astrocytes are substrates for the growth of adult CNS axons in the presence of elevated levels of nerve growth factor. Neuron 7, 1019-1030 (1991).

[3] Philip J. Horner & Fred H. Gage’s accurate review article -from which we are taking some outlines- provides an effective general state of art by the year 2000 about CNS regeneration studies. It may be a good introduction to the field.

Horner, P.J., Gage, F. H., Regenerating the damaged central nervous system, Nature, 407, 963-970, (2000).

[4] Opinions here expressed were extensively discussed with Nicole Cardon and Diane Richmond.