Larry I. Benowitz, PhD
Associate Professor of Neurosurgery
Harvard Medical School/Children's Hospital
Department of Neurosurgery

MRRC Project(s)

R01 EY 05690-23
Molecular Bases of Neuronal Connectivity

Nerve cells in the mature central nervous system (CNS: brain, spinal cord, eye) can not re-establish their connections after injury, nor can intact cells grow new connections to compensate for those that have been lost. As a result of this, victims of traumatic injury, stroke or neurodegenerative diseases suffer permanent and often devastating losses in movement, sensation, bodily functions, and thinking. Recent evidence shows, however, that mature nerve cells can be stimulated to grow new connections under certain circumstances. The goals of the Benowitz lab are to discover the basic mechanisms that control the growth of nerve connections and to apply insights from this work to promote regeneration and functional recovery after CNS injury.

Research Description

Highlights of Major Accomplishments

1. Axon regeneration in the mature optic nerve

2. Functional recovery after stroke using inosine

3. Signaling pathways leading to axon outgrowth

4. mRNA stabilization

1. Axon regeneration in the mature optic nerve

The optic nerve conveys visual information from the eye to the brain. Axons that are injured in the optic nerve normally do not regenerate, and their parent cell bodies, the retinal ganglion cells, soon begin to die. Dr. Yuqin Yin in our lab previously found that by stimulating an inflammatory reaction in the eye, macrophages secrete one or more factors that enable retinal ganglion cells to survive axon damage and regenerate their axons through the optic nerve, a cellular territory that is normally prohibitive to growth. To identify the molecules responsible for this, Dr. Yin developed a cell culture model and discovered that axon regeneration requires 3 components: a low molecular weight factor that is constitutively present in the eye, elevated intracellular cAMP, and a protein that is secreted by macrophages.

Using column chromatography, Drs. Yuqin Yin and Yiming Li isolated the macrophage-derived protein and discovered by mass spectrometric analysis that it is a previously uncharacterirzed growth factor that we refer to as MDP-14. Dr. Yin showed that MDP-14 is a highly potent stimulator or axon regeneration for retinal ganglion cells (1-10 nM), and that its binding to cell surface receptors on retinal ganglion cells requires elevation of intracellular cAMP. In preliminary studies, she has found that MDP-14, when delivered via slow-release microspheres, augments axon regeneration in the mature rat optic nerve. This method may eventually benefit victims of glaucoma and other disorders.

In view of the successful regeneration we are able to obtain in the mature optic nerve, the next question is what genes are involved. To explore this question, Dr. Dietmar Fischer isolated retinal ganglion cells from the numerous other cells that populate the retina by fluorescently labeling the retinal ganglion cells, then using fluorescent-activated cell sorting. mRNA extracted from these cells was amplified, labeled, and applied to microarrays (gene chips). The results of this study showed that, although injury to axons alters the expression of several hundred genes, exposing ganglion cells to macrophage-derived growth factors after axotomy causes only a relatively small number of additional changes in gene expression. We will now investigate which of these genes make the difference between successful regeneration vs. death for retinal ganglion cells.

We have begun to use gene therapy, with adeno-associated virus (AAV) vectors, to investigate the role of various genes on axon regeneration and cell survival. We began by investigating the role of NgR, a receptor on the surface of axons that mediates the inhibitory effects of several myelin proteins on axon growth. Myelin is made by oligodendrocytes and surrounds axons in the central nervous system; 3 myelin proteins that inhibit axon growth exert their effects through the so-called nogo receptor, NgR. We transfected retinal ganglion cells with either a gene to increase levels of NgR or a gene encoding a dominant-negative protein that prevents NgR from working (NgRdn). Increasing levels of NgR completely blocked axon regeneration, whereas expression of NgRdn increased axon regeneration dramatically. Besides demonstrating the critical role of NgR in limiting nerve regeneration in vivo, these results demonstrate that overcoming inhibitory signaling by itself will not lead to extensive regeneration unless neurons' intrinsic growth state is activated. David Goldberg, Sonal Jhaveri and Dietmar Fischer in the lab are beginning to transfer this technology to enhance axon regeneration in the injured spinal cord.

2. Functional recovery after stroke using inosine

The corticospinal tract (CST) carries signals from the cerebral cortex to the spinal cord. The failure of this pathway to regenerate after injury results in devastating losses in voluntary movement of the limbs and digits. Basic research from this lab discovered inosine, a normally occurring molecule that is derived from the metabolism of adenosine, causes nerve cells in culture to regenerate their axons. Inosine was shown to act by passing directly into nerve cells and to activate the cell's program to grow an axon. Because this mechanism appeared from their work to control axon growth in many types of nerve cells, David Goldberg and other members of the lab investigated whether inosine would have a similar effect in the rat's nervous system in vivo. Although the CST is particularly resistant to efforts to get injured pathways to regrow after injury, we found that if we severed it on one side of the brain and then treated the normal, intact side with inosine for 2 weeks, hundreds, and in some cases thousands, of uninjured nerve fibers crossed over from the intact side to the side of the spinal cord which had lost its normal inputs.

In studies carried out by Peng Chen and David Goldberg in the lab, inosine was found to promote the growth of new brain connections and to improve functional outcome after a stroke in the cerebral cortex. After an experimentally created stroke and treatment with insoine, we found, similar to our earlier studies, hundreds of axons growing into denervated areas in the brain and spinal cord. In addition to these anatomical chages, the rats' behavior improved on a number of tasks that are sensitive to cortical integrity. These included the reaching task, the placing reaction and a swimming test. Further studies are defining the "window of opportunity" for inosine treatment, and indicate that treatment must begin within a day or so to be highly effective. In addition, treatment must persist for at least 7 days to obtain the full functional benefits. Current experiments are aimed at discovering ways to enhance inosine's effectiveness. Inosine is expected to enter Phase I/II clinical trials soon on stroke victims, to test safety and effectiveness in enhancing functional recovery.

3. Signaling pathways leading to axon outgrowth

IWe discovered that inosine, a purine nucleoside, passes through the neuron's membrane and activates a "master switch" that controls the expression of genes required for axon growth (e.g., GAP-43, L1, alpha-1 tubulin). Dr. Nina Irwin, Yiming Li, and other members of the Benowitz lab purified N-kinase and discovered that it is related to other protein kinases involved in cellular signaling pathways, but not previously linked to axon growth. Current experiments indicate that the protein that we isolated meets all criteria to be N-kinase, including rapid activation by growth factors, cofactor independence, and importance for axon growth. Dr. Irwin is using microarrays to investigate the pattern of gene expression regulated via N-kinase.

4. mRNA stablilization

A protein called GAP-43, which Dr. Benowitz and other scientists discovered some years back, is important for axon navigation during development and for the ability of nerve cells to reorganize their connections during learning or after injury. In view of the importance of this molecule in controlling the growth state of the nerve cell, Dr. Nina Irwin has investigated the mechanisms that control its levels, using PC12 cells as a model system. The lab previously found that changes in GAP-43 levels that accompany axon growth are controlled in part by regulating the rate at which the mRNA is degraded in the cell. Dr. Irwin found that protein, called ARPP-19, binds to a region of GAP-43 mRNA that controls the stability of the mRNA. Dr. Irwin constructed "reporter" genes, fusing parts of a green fluorescent protein gene with different regions of GAP-43 mRNA; these genes were co-transfected into cells that either contained the ARPP-19 gene in excess or a control gene. ARPP-19 caused the reporter gene linked to the appropriate region of GAP-43 mRNA to be expressed when cells were treated with a growth factor, thus demonstrating a novel mechanism by which growth signals can regulate GAP-43 levels by regulating mRNA stability.

Publications

Schwalb JM, Gu MF, Stuermer CAO, Bastmeyer M, Hu G-f, Boulis NM, Irwin N, Benowitz LI. Optic nerve glia secrete a low molecular weight factor that stimulates retinal ganglion cells to regenerate axons in goldfish. Neuroscience 1996; 72:901-910.

Chao S, Benowitz LI, Krainc D, Irwin N. Use of a two-hybrid system to identify the molecular interactions of GAP-43. Molec Brain Res 1996; 40:195-202.

Perrone-Bizzozero NI, Sower A, Bird ED, Benowitz LI, Ivins KJ, Neve RL. Levels of the growth associated protein GAP-43 are selectively increased in association cortices in schizophrenia. Proc Natl Acad Sci USA 1996; 93:14182-14187.

Benowitz LI, Routtenberg A. GAP-43: an intrinsic determinant of neural development and synaptic plasticity. TINS 1997; 20:84-91.

Irwin N, Baekelandt VL, Goritchenko L, Benowitz LI. Identification of two proteins that bind to a pyrimidine-rich sequence in the 3' untranslated region of GAP-43 mRNA. Nucl Acids Res 1997; 25:1281-1288.

Kawamata T, Dietrich D, Schallert T, Gotts J, Cocke R, Benowitz LI, Finklestein S. Intracisternal basic fibroblast growth factor (bFGF) enhances functional recovery and upregulates the expression of a molecular marker of neuronal sprouting following focal cerebral infarction in the rat. Proc Natl Acad Sci USA 1997; 94:8179-8184.

Benowitz LI, Jing Y, Tabibiazar R, Rosenberg PA, Jo S, Petrausch B, Stuermer C, Irwin N. Axonal regeneration is regulated by an intracellular purine-sensitive mechanism in retinal ganglion cells. J Biol Chem 1998; 273:29626-29634

Madsen JR, MacDonald P, Irwin N, Goldberg DE, Rimm IJ, Stieg PE, Benowitz LI. Tacrolimus (FK-506) improves functional recovery after spinal cord injury in rats. Exper Neurol 1998; 154:673-683.

Jo SA, Wang E, Benowitz LI. CNTF is as an endogenous axogenic factor for mammalian retinal ganglion cells. Neuroscience 1999; 89:579-591.

Benowitz LI, Goldberg D, Madsen JR, Soni D, Irwin N. Inosine stimulates extensive axon collateral growth in the rat corticospinal tract after injury. PNAS USA 1999; 96:13486-13490.

Benowitz LI, Leon S, Tabibiazar R, Jing Y, Irwin N. Axon regeneration in the primary visual pathway of goldfish and rat. In: Ingoglia N, Murray M, editors. Regeneration in the Central Nervous System. NY: Marcel Dekker; 2000.

Leon S, Yin Y, Nguyen J, Irwin N, Benowitz LI. Lens injury stimulates axon regeneration in the mature rat optic nerve. J Neurosci 2000;20:4615-4626.

Petrausch B, Tabibiazar R, Roser T, Jing Y, Goldman D, Stuermer CAO, Irwin N, Benowitz LI. A purine-sensitive pathway regulates multiple genes involved in axon regeneration in goldfish retinal ganglion cells. J Neurosci 2000;20:8031-8041.

Kim HA, Pomeroy SL, Whoriskey W, Pawlitsky I, Benowitz LI, Sicinski P, Stiles CD, Roberts TM. A developmentally regulated switch directs regenerative growth of Schwann cells through cyclin D1. Neuron 2000;26:405-416.

Benowitz LI, Goldberg DE, Irwin N. A purine-sensitive mechanism regulates the molecular program for axon growth. Restor Neurol Neurosci 2001;19(1-2):41-9.

Chen P, Goldberg DE, Kolb B, Lanser M, Benowitz LI. Inosine induces axonal rewiring and improves behavioral outcome after stroke. PNAS 2002;99:9031-9036.

Benowitz LI, Goldberg D, Irwin N. Inosine stimulates axon growth in vitro and in the adult CNS. In: Rossignol S, Doucet G, McKerracher L, editors. Progress in Brain Research; 2002, p. 389-399.

Yin Y, Gui Q, Li Y, Irwin N, Fischer D, Harvey AR, Benowitz LI. Macrophage-derived factors stimulate optic nerve regeneration. J Neurosci 2003;23:2784-2793.

Li Y, Irwin N, Yin Y, Lanser M, Benowitz LI. Axon regeneration in goldfish and rat retinal ganglion cells: differential responsiveness to carbohydrates and cAMP. J Neurosci 2003;23(21):7830-8.

Fischer D, He Z, Benowitz LI. Counteracting the Nogo receptor enhances optic nerve regeneration if retinal ganglion cells are in an active growth state. J Neurosci 2004;24(7):1646-1651.

See Dr. Benowitz's publications via PubMed

Contact Information

E-mail: Larry I. Benowitz, PhD
Associate Professor of Neurosurgery
Harvard Medical School/Children's Hospital
Department of Neurosurgery