Benowitz Lab | Research Projects

Optic nerve regeneration: Inflammation, oncomodulin, and transcriptional control

The inability of neurons to regenerate axons within the CNS has devastating consequences for victims of stroke, spinal cord injury, and other CNS insults. Due to its accessibility, simple anatomy, and functional importance, the optic nerve has been widely studied for insights into positive and negative regulators of CNS regeneration. We discovered that intraocular inflammation enables RGCs, the projection neurons of the eye, to undergo dramatic changes in their transcriptional profile and revert to an active growth state, allowing them to regenerate lengthy axons through the injured optic nerve (Leon et al., 2000; Yin et al., 2003; Fischer et al., 2004). We identified the primary mediator of this phenomenon as Oncomodulin (Ocm), a small Ca2+-binding protein not previously known to have trophic effects, and showed that Ocm binds to a high-affinity receptor on RGCs in a cAMP-dependent manner. Levels of Ocm mRNA and protein increase dramatically in the eye following intraocular inflammation, and, when packaged into slow-release polymer beads together with a cAMP analog, Ocm mimics the regenerative effects of intraocular inflammation (Yin et al., 2006; Yin et al., 2009). Conversely, a peptide antagonist of Ocm blocks inflammation-induced regeneration (Yin et al., 2009). Neutrophils are the primary source of Ocm and are essential forinflammation-induced regeneration (Kurimoto et al., 2013). This work has recently been reviewed (Benowitz and de Lima, 2014).Ongoing projects related to this work are aimed at understanding the cell signaling pathways and sequence of transcriptional changes that underlies successful regeneration in vivo and identifying the receptor for Ocm.

Benowitz LI, de Lima S (2014) Optic Nerve Regeneration. In: The New Visual Neurosciences (Werner JS, Chalupa LM, eds), pp 1387-1406. Cambridge, Massachusetts: The MIT Press.

Fischer D, Petkova V, Thanos S, Benowitz LI (2004) Switching mature retinal ganglion cells to a robust growth state in vivo: gene expression and synergy with RhoA inactivation. J Neurosci 24:8726-8740.

Kurimoto T, Yin Y, Habboub G, Gilbert HY, Li Y, Nakao S, Hafezi-Moghadam A, Benowitz LI (2013) Neutrophils express oncomodulin and promote optic nerve regeneration. J Neurosci 33:14816-14824.

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

Yin Y, Cui Q, Li Y, Irwin N, Fischer D, Harvey AR, Benowitz LI (2003) Macrophage-derived factors stimulate optic nerve regeneration. J Neurosci 23:2284-2293.

Yin Y, Henzl MT, Lorber B, Nakazawa T, Thomas TT, Jiang F, Langer R, Benowitz LI (2006) Oncomodulin is a macrophage-derived signal for axon regeneration in retinal ganglion cells. Nat Neurosci 9:843-852.

Yin Y, Cui Q, Gilbert HY, Yang Y, Yang Z, Berlinicke C, Li Z, Zaverucha-do-Valle C, He H, Petkova V, Zack DJ, Benowitz LI (2009) Oncomodulin links inflammation to optic nerve regeneration. Proc Natl Acad Sci U S A 106:19587-19592

Re-connecting the eye to the brain: combinatorial therapies.

Proteins associated with CNS myelin and the scar that forms at the site of CNS injury inhibit axon growth in culture, yet counteracting the effects of these proteins (e.g., via gene deletion or with antibodies) has generally been insufficient to promote extensive axon regeneration in vivo. Using a gene-therapy approach in the optic nerve model, we found that, although genetic manipulations that rendered RGCs unresponsive to myelin and the glial scar failed to induce extensive axon regeneration after optic nerve injury, these treatments strongly augmented the extent of regeneration induced by intraocular inflammation. These studies reinforced the insufficiency of counteracting inhibitory proteins as a means of promoting CNS regeneration in vivo while at the same time showing the synergistic effects of combining the latter approach with methods that activate neurons’intrinsic growth state (Fischer et al., 2004a; Fischer et al., 2004b). In other studies, we demonstrated a massive synergy between activating RGCs’ growth state via intraocular inflammation and knocking out the gene for pten, a potent cell-intrinsic suppressor of growth (Kurimoto et al., 2010) and showed that this approach enabled some RGCs to regenerate axons the full length of the optic nerve, reinervate the appropriate central target areas in the brain, and restore simple visual responses (de Lima et al., 2012). This represents the first study to demonstrate the possibility of restoring the appropriate circuitry of the visual system after optic nerve damage. Ongoing projects in this area are aimed at developing translationally applicable methods to restore vision after optic nerve injury.

de Lima S, Koriyama Y, Kurimoto T, Oliveira JT, Yin Y, Li Y, Gilbert HY, Fagiolini M, Martinez AM, Benowitz L (2012) Full-length axon regeneration in the adult mouse optic nerve and partial recovery of simple visual behaviors. Proc Natl Acad Sci U S A 109:9149-9154.

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

Fischer D, Petkova V, Thanos S, Benowitz LI (2004b) Switching mature retinal ganglion cells to a robust growth state in vivo: gene expression and synergy with RhoA inactivation. J Neurosci 24:8726-8740.

Kurimoto T, Yin Y, Omura K, Gilbert HY, Kim D, Cen LP, Moko L, Kugler S, Benowitz LI (2010) Long-distance axon regeneration in the mature optic nerve: contributions of oncomodulin, cAMP, and pten gene deletion. J Neurosci 30:15654-15663.

Protection of RGCs in optic nerve injury and glaucoma: Role of zinc and TNF-a.

Glaucoma is a leading cause of blindness worldwide, and although current treatments usually stem the progression of the disease, there are many cases in which this approach is unsuccessful. In two animal models of glaucoma, we elevated intraocular pressure by either episcleral vein cauterization (in rats) or angle closure (in mice) and observed a dramatic elevation of the cytokine TNF-, activation of microglia (Nakazawa et al., 2006; Roh et al., 2012), and a delayed, progressive loss of RGCs, thus mimicking key features of the disease. In one study, we showed that deletion of the gene for TNF- or one of its receptors, or use of an antibody to TNF- greatly slowed or arrested the loss of RGCs despite persistently elevated intraocular pressure (Nakazawa et al., 2006). In the second study, we showed that Etanercept, an FDA-approved, soluble decoy receptor for TNF-, arrested microglial activation at the optic nerve head along and stemmed the loss of RGCs (Roh et al., 2012). These studies have contributed to a growing awareness of the role of TNF- and inflammation as potentially critical factors in the pathophysiology of glaucoma.

In other work, we have found that one of the earliest events to occur in the eye after the optic nerve is injured is a rise in ionic zinc (Zn2+) in the synaptic terminals that amacrine cells make onto the dendrites of RGCs. Within 2 days or so, the Zn2+ accumulates in RGCs. This effect requires the Zn2+transporter ZnT3 and nitric oxide. Chelating Zn2+ leads to the enduring survival of many RGCs and extensive axon regeneration (Li et al., 2014; ms submitted). Ongoing projects in the lab are aimed at understanding the cellular and molecular events that underlie the presynaptic accumulation of zinc after optic nerve injury and the role that zinc plays in suppressing cell survival and axon regeneration.

Li Y, Andereggen L, Omura K, Erdogan B, Asdourian MS, Shrock C, Gilbert H-Y, Yin Y, Apfel UP, Lippard SJ, Rosenberg PA, Benowitz LI (2014) Zinc is a critical regulator of optic nerve regeneration. Program No 39919 2014 Neuroscience Meeting Planner Washington, DC: Society for Neuroscience, 2014.

Nakazawa T, Nakazawa C, Matsubara A, Noda K, Hisatomi T, She H, Michaud N, Hafezi-Moghadam A, Miller JW, Benowitz LI (2006) Tumor necrosis factor-alpha mediates oligodendrocyte death and delayed retinal ganglion cell loss in a mouse model of glaucoma. J Neurosci 26:12633-12641

Roh M, Zhang Y, Murakami Y, Thanos A, Lee SC, Vavvas DG, Benowitz LI, Miller JW (2012) Etanercept, a widely used inhibitor of tumor necrosis factor-alpha (TNF-alpha), prevents retinal ganglion cell loss in a rat model of glaucoma. PLoS One 7:e40065.

Inosine activates the protein kinase Mst3b and enhances recovery after stroke and spinal cord injury.

While investigating why lower vertebrates regenerate their optic nerves, we found that the vitreous and some tissues contain a small molecule that enables goldfish and mouse RGCs to regenerate axons in culture, and identified the principle component as mannose (Schwalb et al., 1995; Li et al., 2003). At the same time, we found that the adenosine metabolite inosine, acting through a direct intracellular mechanism, also stimulates extensive axon outgrowth from RGCs (Benowitz et al., 1998), and went on to identify its target as the protein kinase Mst3b (a splice variant of STK24) and an essential part of the cell-signaling pathway through which many trophic factors promote axon outgrowth in culture and in vivo (Irwin et al., 2006; Lorber et al., 2009). After a unilateral stroke of the rat motor cortex, we found that intraventricular delivery of inosine enhanced the ability of layer 5 pyramidal cells to sprout axon collaterals into the denervated side of the spinal cord and improved skilled use of the impaired forepaw, and that this capacity is strongly enhanced by environmental enrichment or combining inosine with a peptide antagonist of the Nogo receptor (Zai et al., 2009; Zai et al., 2011). Additional studies showed that inosine promotes the formation of “detour circuits” in the spinal cord after transecting the corticospinal tract (CST) at the mid-thoracic level. Inosine enhanced the ability of transected CST axons to sprout collateral branches in the cervical spinal cord that formed synaptic contacts onto spinal interneurons that project from the cervical spinal cord to the lumbar level, thus providing indirect cortical input to the lumbar spinal cord and restoring some volitional control to the hindlimbs (Kim et al., 2013).

Schwalb JM, Boulis NM, Gu MF, Winickoff J, Jackson PS, Irwin N, Benowitz LI (1995) Two factors secreted by the goldfish optic nerve induce retinal ganglion cells to regenerate axons in culture. J Neurosci 15:5514-5525.

Benowitz LI, Jing Y, Tabibiazar R, Jo SA, Petrausch B, Stuermer CA, Rosenberg PA, Irwin N (1998) Axon outgrowth is regulated by an intracellular purine-sensitive mechanism in retinal ganglion cells. J Biol Chem 273:29626-29634.

Li Y, Irwin N, Yin Y, Lanser M, Benowitz LI (2003) Axon regeneration in goldfish and rat retinal ganglion cells: differential responsiveness to carbohydrates and cAMP. J Neurosci 23:7830-7838.

Irwin N, Li YM, O'Toole JE, Benowitz LI (2006) Mst3b, a purine-sensitive Ste20-like protein kinase, regulates axon outgrowth. Proc Natl Acad Sci U S A 103:18320-18325.

Lorber B, Howe ML, Benowitz LI, Irwin N (2009) Mst3b, an Ste20-like kinase, regulates axon regeneration in mature CNS and PNS pathways. Nat Neurosci 12:1407-1414.

Zai L, Ferrari C, Subbaiah S, Havton LA, Coppola G, Strittmatter S, Irwin N, Geschwind D, Benowitz LI (2009) Inosine alters gene expression and axonal projections in neurons contralateral to a cortical infarct and improves skilled use of the impaired limb. J Neurosci 29:8187-8197.

Zai L, Ferrari C, Dice C, Subbaiah S, Havton LA, Coppola G, Geschwind D, Irwin N, Huebner E, Strittmatter SM, Benowitz LI (2011) Inosine augments the effects of a Nogo receptor blocker and of environmental enrichment to restore skilled forelimb use after stroke. J Neurosci 31:5977-5988.

Kim D, Zai L, Liang P, Schaffling C, Ahlborn D, Benowitz LI (2013) Inosine enhances axon sprouting and motor recovery after spinal cord injury. PLoS One 8:e81948.

GAP-43 in axon regeneration and brain plasticity

Unlike mammals, fish and am-phibia can readily regenerate their optic nerves, restore a topographic map of visual space in the brain, and recover vision. Because membrane-associated proteins are transported down axons far more rapidly than cytoskeletal proteins and other compo-nents, we utilized double-isotope labeling and 1- and 2-dimensional gel electrophoresis to identify proteins rapidly-transported proteins that might be involved in axon elongation and in determining the properties of growth cones and early synapses. This work led to the (co)-discovery of the protein that came to be called GAP-43 (Benowitz et al., 1981; Benowitz and Lewis, 1983; Benowitz et al., 1983). We showed that this protein was enriched in the membra-nous fraction of nerve terminals during axon growth and that probes to GAP-43 mRNA and protein provided novel insights into the developmental time-course of the peripheral and central nervous system (Moya et al., 1989; Erzurumlu et al., 1990; Dani et al., 1991; Fitzgerald et al., 1991; Reynolds et al., 1991). We showed that GAP-43 was phosphorylated by a particular iso-form of protein kinase C (Schaechter and Benowitz, 1993), and that it was identical to a protein identified in other contexts as an activity-regulated phosphoprotein in certain synapses (Perrone-Bizzozero et al., 1986). We also showed that, whereas levels of GAP-43 decline in most brain areas in the early postnatal period, in the rat and human brains, it remained highly expressed in the hippocampus and associative areas of cortex, where it may be involved in ongoing structural and/or functional plasticity (Neve et al., 1987; Benowitz et al., 1988; Neve et al., 1988; Benowitz et al., 1989).

Benowitz LI, Shashoua VE, Yoon MG (1981) J Neurosci 1:300-307.

Benowitz LI, Lewis ER (1983) J Neurosci 3:2153-2163.

Benowitz LI, Yoon MG, Lewis ER (1983) Science 222:185-188.

Perrone-Bizzozero NI, Finklestein SP, Benowitz LI (1986) Synthesis of a growth-associated protein by embryonic rat cerebrocortical neurons in vitro. J Neurosci 6:3721-3730.

Neve RL, Perrone-Bizzozero NI, Finklestein S, Zwiers H, Bird E, Kurnit DM, Benowitz LI (1987) The neuronal growth-associated protein GAP-43 (B-50, F1): neuronal specificity, developmental regulation and regional distribution of the human and rat mRNAs. Brain Res 388:177-183.Neve RL,

Finch EA, Bird ED, Benowitz LI (1988) Growth-associated protein GAP-43 is expressed selectively in associative regions of the adult human brain. Proc Natl Acad Sci USA 85:3638-3642.

Benowitz LI, Apostolides PJ, Perrone-Bizzozero N, Finklestein SP, Zwiers H (1988) J Neurosci 8:339-352.

Benowitz LI, Perrone-Bizzozero NI, Finklestein SP, Bird ED (1989) J Neurosci 9:990-995.

Moya KL, Jhaveri S, Schneider GE, Benowitz LI (1989) Immunohistochemical localization of GAP-43 in the developing hamster retinofugal pathway. Journal of Comparative Neurology 288:51-58.

Erzurumlu RS, Jhaveri S, Benowitz LI (1990) Journal of Comparative Neurology 292:443-456.

Dani JW, Armstrong DM, Benowitz LI (1991) Neuroscience 40:277-287.

Fitzgerald M, Reynolds ML, Benowitz LI (1991) Neuroscience 41:187-199.

Reynolds ML, Fitzgerald M, Benowitz LI (1991) GAP-43 expression in developing cutaneous and muscle nerves in the rat hindlimb. Neuroscience 41:201-211.

Schaechter JD, Benowitz LI (1993) Activation of protein kinase C by arachidonic acid selectively enhances the phosphorylation of GAP-43 in nerve terminal membranes. J Neurosci 13:4361-4371.