The inability of retinal ganglion cells (RGCs) to regenerate damaged axons through the optic nerv... more The inability of retinal ganglion cells (RGCs) to regenerate damaged axons through the optic nerve has dire consequences for victims of traumatic nerve injury and certain neurodegenerative diseases. Several strategies have been shown to induce appreciable regeneration in vivo, but the regrowth of axons through the entire optic nerve and on into the brain remains a major challenge. We show here that the induction of a controlled inflammatory response in the eye, when combined with elevation of intracellular cAMP and deletion of the gene encoding pten (phosphatase and tensin homolog), enables RGCs to regenerate axons the full length of the optic nerve in mature mice; approximately half of these axons cross the chiasm, and a rare subset (ϳ1%) manages to enter the thalamus. Consistent with our previous findings, the axon-promoting effects of inflammation were shown to require the macrophage-derived growth factor Oncomodulin (Ocm). Elevation of cAMP increased the ability of Ocm to bind to its receptors in the inner retina and augmented inflammation-induced regeneration twofold. Inflammation combined with elevated cAMP and PTEN deletion increased activation of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase signaling pathways and augmented regeneration ϳ10-fold over the level induced by either pten deletion or Zymosan alone. Thus, treatments that synergistically alter the intrinsic growth state of RGCs produce unprecedented levels of axon regeneration in the optic nerve, a CNS pathway long believed to be incapable of supporting such growth.
Despite years of research, no effective therapy is yet available for the treatment of traumatic b... more Despite years of research, no effective therapy is yet available for the treatment of traumatic brain injury (TBI). The most prevalent and debilitating features in survivors of TBI are cognitive deficits and motor dysfunction. A potential therapeutic method for improving the function of patients following TBI would be to restore, at least in part, plasticity to the CNS in a controlled way that would allow for the formation of compensatory circuits. Inosine, a naturally occurring purine nucleoside, has been shown to promote axon collateral growth in the corticospinal tract (CST) following stroke and focal TBI. In the present study, we investigated the effects of inosine on motor and cognitive deficits, CST sprouting, and expression of synaptic proteins in an experimental model of closed head injury (CHI). Treatment with inosine (100 mg/kg i.p. at 1, 24 and 48 h following CHI) improved outcome after TBI, significantly decreasing the neurological severity score (NSS, po0.04 vs. saline), an aggregate measure of performance on several tasks. It improved non-spatial cognitive performance (object recognition, po0.016 vs. saline) but had little effect on sensorimotor coordination (rotarod) and spatial cognitive functions (Y-maze). Inosine did not affect CST sprouting in the lumbar spinal cord but did restore levels of the growth-associated protein GAP-43 in the hippocampus, though not in the cerebral cortex. Our results suggest that inosine may improve functional outcome after TBI.
Sir, we wish to comment on the paper ‘Astrocyte-derived CNTF switches mature retinal ganglion cel... more Sir, we wish to comment on the paper ‘Astrocyte-derived CNTF switches mature retinal ganglion cells (RGCs) to a regenerative state following inflammatory stimulation’ (Muller et al., 2007). RGCs, the projection neurons of the eye, are normally unable to regenerate their axons if the optic nerve is injured. However, they can be stimulated to do so by inducing an inflammatory reaction in the eye (Leon et al., 2000; Yin et al., 2003). The evidence presented later argues against CNTF playing a central role in this transformation. The authors do not demonstrate that CNTF can directly stimulate RGCs to regenerate axons. They show that co-culturing retinal explants with lens or zymosan increases CNTF expression in retinal glia, but they do not show that this CNTF is secreted nor whether this stimulation, or even
Gene therapy currently is one of the most promising treatments for genetic blindness. With the su... more Gene therapy currently is one of the most promising treatments for genetic blindness. With the success of the phase I/ II trials for Leber’s congenital amaurosis (LCA) RPE65 and choroideremia (CHM), gene transfer to the retina has been shown to be technically safe, and with the potential to lead to efficacy in the treatment of retinal forms of blindness through gene augmentation in recessive or X-linked disease (MacLaren et al., 2014; Simonelli et al., 2010). A multitude of single gene disorders including retinitis pigmentosa (RP), caused by defects in over 60 different genes, remain. The challenges to bring this technology to patients in the wide spectrum of blinding disorders were discussed. There are several technical and logistical issues that are seen as hurdles in the development path that are either specific to particular approaches/indications or shared among many.
Retinal cell death is the leading cause of vision loss in the majority of blinding conditions. Re... more Retinal cell death is the leading cause of vision loss in the majority of blinding conditions. Research focused on understanding the mechanism by which retinal cells die provides the cornerstone on which we will build and develop therapies meant to target those mechanisms. Neuroprotective therapies are one approach that is aimed at preventing, slowing, and ultimately, reversing the neurodegenerative processes that drive retinal diseases.
Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for several ocular diseases... more Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for several ocular diseases and induces optic nerve regeneration in animal models. Paradoxically, however, although CNTF gene therapy promotes extensive regeneration, recombinant CNTF (rCNTF) has little effect. Because intraocular viral vectors induce inflammation, and because CNTF is an immune modulator, we investigated whether CNTF gene therapy acts indirectly through other immune mediators. The beneficial effects of CNTF gene therapy remained unchanged after deleting CNTF receptor alpha (CNTFRα) in retinal ganglion cells (RGCs), the projection neurons of the retina, but were diminished by depleting neutrophils or by genetically suppressing monocyte infiltration. CNTF gene therapy increased expression of C-C motif chemokine ligand 5 (CCL5) in immune cells and retinal glia, and recombinant CCL5 induced extensive axon regeneration. Conversely, CRISPR-mediated knockdown of the cognate receptor (CCR5) in RGCs or tre...
The inability of neurons to regenerate long axons within the CNS is a major impediment to improvi... more The inability of neurons to regenerate long axons within the CNS is a major impediment to improving outcome after spinal cord injury, stroke, and other CNS insults. Recent advances have uncovered an intrinsic program that involves coordinate regulation by multiple transcription factors that can be manipulated to enhance growth in the peripheral nervous system. Here, we used a system-genomics approach to characterize regulatory relationships of regeneration-associated transcription factors, identifying RE1-Silencing Transcription Factor (REST; Neuron-Restrictive Silencer Factor, NRSF) as a predicted upstream suppressor of a pro-regenerative gene program associated with axon regeneration in the CNS. We validate our predictions using multiple paradigms, showing that mature mice bearing cell type-specific deletions of REST or expressing dominant-negative mutant REST showed improved regeneration of the corticospinal tract and optic nerve, accompanied by upregulation of regeneration-assoc...
Growth cones (GCs) are structures associated with growing neurons. GC membrane expansion, which n... more Growth cones (GCs) are structures associated with growing neurons. GC membrane expansion, which necessitates protein-lipid interactions, is critical to axonal elongation in development and in adult neuritogenesis. We present a multi-omic analysis that integrates proteomics and lipidomics data for the identification of GC pathways, cell phenotypes, and lipid-protein interactions, with an analytic platform to facilitate the visualization of these data. We combine lipidomic data from GC and adult axonal regeneration following optic nerve crush. Our results reveal significant molecular variability in GCs across developmental ages that aligns with the upregulation and downregulation of lipid metabolic processes and correlates with distinct changes in the lipid composition of GC plasmalemma. We find that these processes also define the transition into a growth-permissive state in the adult central nervous system. The insight derived from these analyses will aid in promoting adult regeneration and functional innervation in devastating neurodegenerative diseases.
SUMMARYRetinal ganglion cell (RGC) death is the hallmark of glaucoma. Axonal injury is thought to... more SUMMARYRetinal ganglion cell (RGC) death is the hallmark of glaucoma. Axonal injury is thought to precede RGC loss in glaucoma, and thus studies using an optic nerve (ON) crush model have been widely used to investigate mechanisms of cell death that are common to both conditions. Prior work has focused on the involvement of caspases in RGC death, but little is known about the contribution of other forms of cell death such as necrosis. In this study we show that receptor interacting protein (RIP) kinase-mediated necrosis normally plays a role in RGC death and acts in concert with caspase-dependent apoptosis. The expression of RIP3, a key activator of RIP1 kinase, as well as caspase activity, increased following ON injury. Caspase inhibition alone failed to provide substantial protection to injured RGCs and unexpectedly exacerbated necrosis. In contrast, pharmacologic or genetic inhibition of RIP kinases in combination with caspase blockade delayed both apoptotic and necrotic RGC deat...
Ischemic stroke (IS) is a leading disease with high mortality and disability, as well as with lim... more Ischemic stroke (IS) is a leading disease with high mortality and disability, as well as with limited therapeutic window. Biomarkers for earlier diagnosis of IS have long been pursued. Family and twin studies confirm that genetic variations play an important role in IS pathogenesis. Besides DNA mutations found previously by genetic linkage analysis for monogenic IS (Mendelian inheritance), recent studies using genome-wide associated study (GWAS) and microRNA expression profiling have resulted in a large number of DNA and microRNA biomarkers in polygenic IS (sporadic IS), especially in different IS subtypes and imaging phenotypes. The present review summarizes genetic markers discovered by clinical studies and discusses their pathogenic molecular mechanisms involved in developmental or regenerative anomalies of blood vessel walls, neuronal apoptosis, excitotoxic death, inflammation, neurogenesis, and angiogenesis. The possible impact of environment on genetics is addressed as well. W...
The inability of axons to regenerate over long-distances in the central nervous system (CNS) limi... more The inability of axons to regenerate over long-distances in the central nervous system (CNS) limits the recovery of sensory, motor, and cognitive functions after various CNS injuries and diseases. Although pre-clinical studies have identified a number of manipulations that stimulate some degree of axon growth after CNS damage, the extent of recovery remains quite limited, emphasizing the need for improved therapies. Here, we used traumatic injury to the mouse optic nerve as a model system to test the effects of combining several treatments that have recently been found to promote axon regeneration without the risks associated with manipulating known tumor suppressors or oncogenes. The treatments tested here include TPEN, a chelator of mobile (free) zinc (Zn2+); shRNA against the axon growth-suppressing transcription factor Klf9; and the atypical growth factor oncomodulin combined with a cAMP analog. Whereas some combinatorial treatments produced only marginally stronger effects than...
International Journal of Developmental Neuroscience
spinal cord explants were cultured in collagen gel for a few days, axons from the explant exhibit... more spinal cord explants were cultured in collagen gel for a few days, axons from the explant exhibited radial outgrowth into the matrix. However, the explants were co-cultured with the notochord explants, growing Cnt1-positive spinal axons were repelled by notochord-derived repulsive activity. This repulsive activity was abolished by the addition of either a monoclonal anti-Cnt1 antibody, chondroitin sulfate A or chondroitinase ABC to the culture medium. An antibody that recognizes chondroitin sulfate A and C labeled immunohistochemically the notochord in embryo sections and the collagen gel matrix around the cultured notochord explant. Addition of chondroitinase ABC into the culture eliminated the immunoreactivity in the gel matrix. These results suggest that the notochord-derived chondroitin sulfate proteoglycan acts as a repulsive signaling molecule that is recognized by Cnt1 on spinal sensory axons. This guidance mechanism would be involved in navigation of the growing sensory axons not to enter mid-body areas where the notochord exist, resulting in their subepidermal extension.
This review arose from a discussion of regenerative therapies to treat optic nerve degeneration i... more This review arose from a discussion of regenerative therapies to treat optic nerve degeneration in glaucoma at the 2015 Lasker/IRRF Initiative on Astrocytes and Glaucomatous Neurodegeneration. In addition to the authors, participants included Jonathan Crowston, Andrew Huberman, Elaine Johnson, Richard Lu, Hemai Phatnami, Rebecca Sappington, and Don Zack. Glaucoma is a neurodegenerative disease of the optic nerve, and is the leading cause of irreversible blindness worldwide. The disease progresses as sensitivity to intraocular pressure (IOP) is conveyed through the optic nerve head to distal retinal ganglion cell (RGC) projections. Because the nerve and retina are components of the central nervous system (CNS), their intrinsic regenerative capacity is limited. However, recent research in regenerative therapies has resulted in multiple breakthroughs that may unlock the optic nerve's regenerative potential. Increasing levels of Schwann-cell derived trophic factors and reducing potent cell-intrinsic suppressors of regeneration have resulted in axonal regeneration even beyond the optic chiasm. Despite this success, many challenges remain. RGC axons must be able to form new connections with their appropriate targets in central brain regions and these connections must be retinotopically correct. Furthermore, for new axons penetrating the optic projection, oligodendrocyte glia must provide myelination. Additionally, reactive gliosis and inflammation that increase the regenerative capacity must be outweigh pro-apoptotic processes to create an environment within which maximal regeneration can occur.
Proceedings of the National Academy of Sciences of the United States of America, Jan 10, 2017
Retinal ganglion cells (RGCs), the projection neurons of the eye, cannot regenerate their axons o... more Retinal ganglion cells (RGCs), the projection neurons of the eye, cannot regenerate their axons once the optic nerve has been injured and soon begin to die. Whereas RGC death and regenerative failure are widely viewed as being cell-autonomous or influenced by various types of glia, we report here that the dysregulation of mobile zinc (Zn(2+)) in retinal interneurons is a primary factor. Within an hour after the optic nerve is injured, Zn(2+) increases several-fold in retinal amacrine cell processes and continues to rise over the first day, then transfers slowly to RGCs via vesicular release. Zn(2+) accumulation in amacrine cell processes involves the Zn(2+) transporter protein ZnT-3, and deletion of slc30a3, the gene encoding ZnT-3, promotes RGC survival and axon regeneration. Intravitreal injection of Zn(2+) chelators enables many RGCs to survive for months after nerve injury and regenerate axons, and enhances the prosurvival and regenerative effects of deleting the gene for phosph...
Inosine, a naturally occurring purine nucleoside, has been shown to stimulate axonal growth in ce... more Inosine, a naturally occurring purine nucleoside, has been shown to stimulate axonal growth in cell culture and promote corticospinal tract axons to sprout collateral branches after stroke, spinal cord injury and TBI in rodent models. To explore the effects of inosine on the recovery of motor function following cortical injury in the rhesus monkey. After being trained on a test of fine motor function of the hand, monkeys received a lesion limited to the area of the hand representation in primary motor cortex. Beginning 24 hours after this injury and continuing daily thereafter, monkeys received orally administered inosine (500 mg) or placebo. Retesting of motor function began on the 14th day after injury and continued for 12 weeks. During the first 14 days after surgery, there was evidence of significant recovery within the inosine-treated group on measures of fine motor function of the hand, measures of hand strength and digit flexion. While there was no effect of treatment on the time to retrieve a reward, the treated monkeys returned to asymptotic levels of grasp performance significantly faster than the untreated monkeys. Additionally, the treated monkeys evidenced a greater degree of recovery in terms of maturity of grasp pattern. These findings demonstrate that inosine can enhance recovery of function following cortical injury in monkeys.
The Journal of neuroscience : the official journal of the Society for Neuroscience, Aug 31, 2016
Action potential initiation and propagation in myelinated axons require ion channel clustering at... more Action potential initiation and propagation in myelinated axons require ion channel clustering at axon initial segments (AIS) and nodes of Ranvier. Disruption of these domains after injury impairs nervous system function. Traditionally, injured CNS axons are considered refractory to regeneration, but some recent approaches challenge this view by showing robust long-distance regeneration. However, whether these approaches allow remyelination and promote the reestablishment of AIS and nodes of Ranvier is unknown. Using mouse optic nerve crush as a model for CNS traumatic injury, we performed a detailed analysis of AIS and node disruption after nerve crush. We found significant disruption of AIS and loss of nodes within days of the crush, and complete loss of nodes 1 week after injury. Genetic deletion of the tumor suppressor phosphatase and tensin homolog (Pten) in retinal ganglion cells (RGCs), coupled with stimulation of RGCs by inflammation and cAMP, dramatically enhanced regenerat...
The inability of retinal ganglion cells (RGCs) to regenerate damaged axons through the optic nerv... more The inability of retinal ganglion cells (RGCs) to regenerate damaged axons through the optic nerve has dire consequences for victims of traumatic nerve injury and certain neurodegenerative diseases. Several strategies have been shown to induce appreciable regeneration in vivo, but the regrowth of axons through the entire optic nerve and on into the brain remains a major challenge. We show here that the induction of a controlled inflammatory response in the eye, when combined with elevation of intracellular cAMP and deletion of the gene encoding pten (phosphatase and tensin homolog), enables RGCs to regenerate axons the full length of the optic nerve in mature mice; approximately half of these axons cross the chiasm, and a rare subset (ϳ1%) manages to enter the thalamus. Consistent with our previous findings, the axon-promoting effects of inflammation were shown to require the macrophage-derived growth factor Oncomodulin (Ocm). Elevation of cAMP increased the ability of Ocm to bind to its receptors in the inner retina and augmented inflammation-induced regeneration twofold. Inflammation combined with elevated cAMP and PTEN deletion increased activation of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase signaling pathways and augmented regeneration ϳ10-fold over the level induced by either pten deletion or Zymosan alone. Thus, treatments that synergistically alter the intrinsic growth state of RGCs produce unprecedented levels of axon regeneration in the optic nerve, a CNS pathway long believed to be incapable of supporting such growth.
Despite years of research, no effective therapy is yet available for the treatment of traumatic b... more Despite years of research, no effective therapy is yet available for the treatment of traumatic brain injury (TBI). The most prevalent and debilitating features in survivors of TBI are cognitive deficits and motor dysfunction. A potential therapeutic method for improving the function of patients following TBI would be to restore, at least in part, plasticity to the CNS in a controlled way that would allow for the formation of compensatory circuits. Inosine, a naturally occurring purine nucleoside, has been shown to promote axon collateral growth in the corticospinal tract (CST) following stroke and focal TBI. In the present study, we investigated the effects of inosine on motor and cognitive deficits, CST sprouting, and expression of synaptic proteins in an experimental model of closed head injury (CHI). Treatment with inosine (100 mg/kg i.p. at 1, 24 and 48 h following CHI) improved outcome after TBI, significantly decreasing the neurological severity score (NSS, po0.04 vs. saline), an aggregate measure of performance on several tasks. It improved non-spatial cognitive performance (object recognition, po0.016 vs. saline) but had little effect on sensorimotor coordination (rotarod) and spatial cognitive functions (Y-maze). Inosine did not affect CST sprouting in the lumbar spinal cord but did restore levels of the growth-associated protein GAP-43 in the hippocampus, though not in the cerebral cortex. Our results suggest that inosine may improve functional outcome after TBI.
Sir, we wish to comment on the paper ‘Astrocyte-derived CNTF switches mature retinal ganglion cel... more Sir, we wish to comment on the paper ‘Astrocyte-derived CNTF switches mature retinal ganglion cells (RGCs) to a regenerative state following inflammatory stimulation’ (Muller et al., 2007). RGCs, the projection neurons of the eye, are normally unable to regenerate their axons if the optic nerve is injured. However, they can be stimulated to do so by inducing an inflammatory reaction in the eye (Leon et al., 2000; Yin et al., 2003). The evidence presented later argues against CNTF playing a central role in this transformation. The authors do not demonstrate that CNTF can directly stimulate RGCs to regenerate axons. They show that co-culturing retinal explants with lens or zymosan increases CNTF expression in retinal glia, but they do not show that this CNTF is secreted nor whether this stimulation, or even
Gene therapy currently is one of the most promising treatments for genetic blindness. With the su... more Gene therapy currently is one of the most promising treatments for genetic blindness. With the success of the phase I/ II trials for Leber’s congenital amaurosis (LCA) RPE65 and choroideremia (CHM), gene transfer to the retina has been shown to be technically safe, and with the potential to lead to efficacy in the treatment of retinal forms of blindness through gene augmentation in recessive or X-linked disease (MacLaren et al., 2014; Simonelli et al., 2010). A multitude of single gene disorders including retinitis pigmentosa (RP), caused by defects in over 60 different genes, remain. The challenges to bring this technology to patients in the wide spectrum of blinding disorders were discussed. There are several technical and logistical issues that are seen as hurdles in the development path that are either specific to particular approaches/indications or shared among many.
Retinal cell death is the leading cause of vision loss in the majority of blinding conditions. Re... more Retinal cell death is the leading cause of vision loss in the majority of blinding conditions. Research focused on understanding the mechanism by which retinal cells die provides the cornerstone on which we will build and develop therapies meant to target those mechanisms. Neuroprotective therapies are one approach that is aimed at preventing, slowing, and ultimately, reversing the neurodegenerative processes that drive retinal diseases.
Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for several ocular diseases... more Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for several ocular diseases and induces optic nerve regeneration in animal models. Paradoxically, however, although CNTF gene therapy promotes extensive regeneration, recombinant CNTF (rCNTF) has little effect. Because intraocular viral vectors induce inflammation, and because CNTF is an immune modulator, we investigated whether CNTF gene therapy acts indirectly through other immune mediators. The beneficial effects of CNTF gene therapy remained unchanged after deleting CNTF receptor alpha (CNTFRα) in retinal ganglion cells (RGCs), the projection neurons of the retina, but were diminished by depleting neutrophils or by genetically suppressing monocyte infiltration. CNTF gene therapy increased expression of C-C motif chemokine ligand 5 (CCL5) in immune cells and retinal glia, and recombinant CCL5 induced extensive axon regeneration. Conversely, CRISPR-mediated knockdown of the cognate receptor (CCR5) in RGCs or tre...
The inability of neurons to regenerate long axons within the CNS is a major impediment to improvi... more The inability of neurons to regenerate long axons within the CNS is a major impediment to improving outcome after spinal cord injury, stroke, and other CNS insults. Recent advances have uncovered an intrinsic program that involves coordinate regulation by multiple transcription factors that can be manipulated to enhance growth in the peripheral nervous system. Here, we used a system-genomics approach to characterize regulatory relationships of regeneration-associated transcription factors, identifying RE1-Silencing Transcription Factor (REST; Neuron-Restrictive Silencer Factor, NRSF) as a predicted upstream suppressor of a pro-regenerative gene program associated with axon regeneration in the CNS. We validate our predictions using multiple paradigms, showing that mature mice bearing cell type-specific deletions of REST or expressing dominant-negative mutant REST showed improved regeneration of the corticospinal tract and optic nerve, accompanied by upregulation of regeneration-assoc...
Growth cones (GCs) are structures associated with growing neurons. GC membrane expansion, which n... more Growth cones (GCs) are structures associated with growing neurons. GC membrane expansion, which necessitates protein-lipid interactions, is critical to axonal elongation in development and in adult neuritogenesis. We present a multi-omic analysis that integrates proteomics and lipidomics data for the identification of GC pathways, cell phenotypes, and lipid-protein interactions, with an analytic platform to facilitate the visualization of these data. We combine lipidomic data from GC and adult axonal regeneration following optic nerve crush. Our results reveal significant molecular variability in GCs across developmental ages that aligns with the upregulation and downregulation of lipid metabolic processes and correlates with distinct changes in the lipid composition of GC plasmalemma. We find that these processes also define the transition into a growth-permissive state in the adult central nervous system. The insight derived from these analyses will aid in promoting adult regeneration and functional innervation in devastating neurodegenerative diseases.
SUMMARYRetinal ganglion cell (RGC) death is the hallmark of glaucoma. Axonal injury is thought to... more SUMMARYRetinal ganglion cell (RGC) death is the hallmark of glaucoma. Axonal injury is thought to precede RGC loss in glaucoma, and thus studies using an optic nerve (ON) crush model have been widely used to investigate mechanisms of cell death that are common to both conditions. Prior work has focused on the involvement of caspases in RGC death, but little is known about the contribution of other forms of cell death such as necrosis. In this study we show that receptor interacting protein (RIP) kinase-mediated necrosis normally plays a role in RGC death and acts in concert with caspase-dependent apoptosis. The expression of RIP3, a key activator of RIP1 kinase, as well as caspase activity, increased following ON injury. Caspase inhibition alone failed to provide substantial protection to injured RGCs and unexpectedly exacerbated necrosis. In contrast, pharmacologic or genetic inhibition of RIP kinases in combination with caspase blockade delayed both apoptotic and necrotic RGC deat...
Ischemic stroke (IS) is a leading disease with high mortality and disability, as well as with lim... more Ischemic stroke (IS) is a leading disease with high mortality and disability, as well as with limited therapeutic window. Biomarkers for earlier diagnosis of IS have long been pursued. Family and twin studies confirm that genetic variations play an important role in IS pathogenesis. Besides DNA mutations found previously by genetic linkage analysis for monogenic IS (Mendelian inheritance), recent studies using genome-wide associated study (GWAS) and microRNA expression profiling have resulted in a large number of DNA and microRNA biomarkers in polygenic IS (sporadic IS), especially in different IS subtypes and imaging phenotypes. The present review summarizes genetic markers discovered by clinical studies and discusses their pathogenic molecular mechanisms involved in developmental or regenerative anomalies of blood vessel walls, neuronal apoptosis, excitotoxic death, inflammation, neurogenesis, and angiogenesis. The possible impact of environment on genetics is addressed as well. W...
The inability of axons to regenerate over long-distances in the central nervous system (CNS) limi... more The inability of axons to regenerate over long-distances in the central nervous system (CNS) limits the recovery of sensory, motor, and cognitive functions after various CNS injuries and diseases. Although pre-clinical studies have identified a number of manipulations that stimulate some degree of axon growth after CNS damage, the extent of recovery remains quite limited, emphasizing the need for improved therapies. Here, we used traumatic injury to the mouse optic nerve as a model system to test the effects of combining several treatments that have recently been found to promote axon regeneration without the risks associated with manipulating known tumor suppressors or oncogenes. The treatments tested here include TPEN, a chelator of mobile (free) zinc (Zn2+); shRNA against the axon growth-suppressing transcription factor Klf9; and the atypical growth factor oncomodulin combined with a cAMP analog. Whereas some combinatorial treatments produced only marginally stronger effects than...
International Journal of Developmental Neuroscience
spinal cord explants were cultured in collagen gel for a few days, axons from the explant exhibit... more spinal cord explants were cultured in collagen gel for a few days, axons from the explant exhibited radial outgrowth into the matrix. However, the explants were co-cultured with the notochord explants, growing Cnt1-positive spinal axons were repelled by notochord-derived repulsive activity. This repulsive activity was abolished by the addition of either a monoclonal anti-Cnt1 antibody, chondroitin sulfate A or chondroitinase ABC to the culture medium. An antibody that recognizes chondroitin sulfate A and C labeled immunohistochemically the notochord in embryo sections and the collagen gel matrix around the cultured notochord explant. Addition of chondroitinase ABC into the culture eliminated the immunoreactivity in the gel matrix. These results suggest that the notochord-derived chondroitin sulfate proteoglycan acts as a repulsive signaling molecule that is recognized by Cnt1 on spinal sensory axons. This guidance mechanism would be involved in navigation of the growing sensory axons not to enter mid-body areas where the notochord exist, resulting in their subepidermal extension.
This review arose from a discussion of regenerative therapies to treat optic nerve degeneration i... more This review arose from a discussion of regenerative therapies to treat optic nerve degeneration in glaucoma at the 2015 Lasker/IRRF Initiative on Astrocytes and Glaucomatous Neurodegeneration. In addition to the authors, participants included Jonathan Crowston, Andrew Huberman, Elaine Johnson, Richard Lu, Hemai Phatnami, Rebecca Sappington, and Don Zack. Glaucoma is a neurodegenerative disease of the optic nerve, and is the leading cause of irreversible blindness worldwide. The disease progresses as sensitivity to intraocular pressure (IOP) is conveyed through the optic nerve head to distal retinal ganglion cell (RGC) projections. Because the nerve and retina are components of the central nervous system (CNS), their intrinsic regenerative capacity is limited. However, recent research in regenerative therapies has resulted in multiple breakthroughs that may unlock the optic nerve's regenerative potential. Increasing levels of Schwann-cell derived trophic factors and reducing potent cell-intrinsic suppressors of regeneration have resulted in axonal regeneration even beyond the optic chiasm. Despite this success, many challenges remain. RGC axons must be able to form new connections with their appropriate targets in central brain regions and these connections must be retinotopically correct. Furthermore, for new axons penetrating the optic projection, oligodendrocyte glia must provide myelination. Additionally, reactive gliosis and inflammation that increase the regenerative capacity must be outweigh pro-apoptotic processes to create an environment within which maximal regeneration can occur.
Proceedings of the National Academy of Sciences of the United States of America, Jan 10, 2017
Retinal ganglion cells (RGCs), the projection neurons of the eye, cannot regenerate their axons o... more Retinal ganglion cells (RGCs), the projection neurons of the eye, cannot regenerate their axons once the optic nerve has been injured and soon begin to die. Whereas RGC death and regenerative failure are widely viewed as being cell-autonomous or influenced by various types of glia, we report here that the dysregulation of mobile zinc (Zn(2+)) in retinal interneurons is a primary factor. Within an hour after the optic nerve is injured, Zn(2+) increases several-fold in retinal amacrine cell processes and continues to rise over the first day, then transfers slowly to RGCs via vesicular release. Zn(2+) accumulation in amacrine cell processes involves the Zn(2+) transporter protein ZnT-3, and deletion of slc30a3, the gene encoding ZnT-3, promotes RGC survival and axon regeneration. Intravitreal injection of Zn(2+) chelators enables many RGCs to survive for months after nerve injury and regenerate axons, and enhances the prosurvival and regenerative effects of deleting the gene for phosph...
Inosine, a naturally occurring purine nucleoside, has been shown to stimulate axonal growth in ce... more Inosine, a naturally occurring purine nucleoside, has been shown to stimulate axonal growth in cell culture and promote corticospinal tract axons to sprout collateral branches after stroke, spinal cord injury and TBI in rodent models. To explore the effects of inosine on the recovery of motor function following cortical injury in the rhesus monkey. After being trained on a test of fine motor function of the hand, monkeys received a lesion limited to the area of the hand representation in primary motor cortex. Beginning 24 hours after this injury and continuing daily thereafter, monkeys received orally administered inosine (500 mg) or placebo. Retesting of motor function began on the 14th day after injury and continued for 12 weeks. During the first 14 days after surgery, there was evidence of significant recovery within the inosine-treated group on measures of fine motor function of the hand, measures of hand strength and digit flexion. While there was no effect of treatment on the time to retrieve a reward, the treated monkeys returned to asymptotic levels of grasp performance significantly faster than the untreated monkeys. Additionally, the treated monkeys evidenced a greater degree of recovery in terms of maturity of grasp pattern. These findings demonstrate that inosine can enhance recovery of function following cortical injury in monkeys.
The Journal of neuroscience : the official journal of the Society for Neuroscience, Aug 31, 2016
Action potential initiation and propagation in myelinated axons require ion channel clustering at... more Action potential initiation and propagation in myelinated axons require ion channel clustering at axon initial segments (AIS) and nodes of Ranvier. Disruption of these domains after injury impairs nervous system function. Traditionally, injured CNS axons are considered refractory to regeneration, but some recent approaches challenge this view by showing robust long-distance regeneration. However, whether these approaches allow remyelination and promote the reestablishment of AIS and nodes of Ranvier is unknown. Using mouse optic nerve crush as a model for CNS traumatic injury, we performed a detailed analysis of AIS and node disruption after nerve crush. We found significant disruption of AIS and loss of nodes within days of the crush, and complete loss of nodes 1 week after injury. Genetic deletion of the tumor suppressor phosphatase and tensin homolog (Pten) in retinal ganglion cells (RGCs), coupled with stimulation of RGCs by inflammation and cAMP, dramatically enhanced regenerat...
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Papers by Larry Benowitz