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Peripheral nervous system genes expressed in central neurons induce growth on inhibitory substrates.

Buchser WJ, Smith RP, Pardinas JR, Haddox CL, Hutson T, Moon L, Hoffman SR, Bixby JL, Lemmon VP - PLoS ONE (2012)

Bottom Line: Peripheral nervous system (PNS) neurons exhibit increased regenerative ability compared to central nervous system neurons, even in the presence of inhibitory environments.Several known growth associated proteins potentiated neurite growth on laminin.Bioinformatic approaches also uncovered a number of novel gene families that altered neurite growth of CNS neurons.

View Article: PubMed Central - PubMed

Affiliation: Miami Project to Cure Paralysis, Department of Pharmacology, University of Miami, Miller School of Medicine, Miami, Florida, United States of America.

ABSTRACT
Trauma to the spinal cord and brain can result in irreparable loss of function. This failure of recovery is in part due to inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans (CSPGs). Peripheral nervous system (PNS) neurons exhibit increased regenerative ability compared to central nervous system neurons, even in the presence of inhibitory environments. Previously, we identified over a thousand genes differentially expressed in PNS neurons relative to CNS neurons. These genes represent intrinsic differences that may account for the PNS's enhanced regenerative ability. Cerebellar neurons were transfected with cDNAs for each of these PNS genes to assess their ability to enhance neurite growth on inhibitory (CSPG) or permissive (laminin) substrates. Using high content analysis, we evaluated the phenotypic profile of each neuron to extract meaningful data for over 1100 genes. Several known growth associated proteins potentiated neurite growth on laminin. Most interestingly, novel genes were identified that promoted neurite growth on CSPGs (GPX3, EIF2B5, RBMX). Bioinformatic approaches also uncovered a number of novel gene families that altered neurite growth of CNS neurons.

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Active growth genes in cerebellar neurons and their expression pattern.A, B, Phenotypic results of overexpression in cerebellar granule neurons (CGNs) confirmed by four replicates following the primary screen. Bars represent normalized values, centered on the neutral control, mCherry. Three parameters: number of branches (gray), neurite total length (white) and primary neurite count (black), are reported for transfected neurons (GFP positive). Significant results are indicated with asterisks (ANOVA). Genes had effects on CSPG (A), laminin (B), or both substrates. mCherry transfection with Gö6976, the positive control, is plotted on the far right. C–F, Representative images of CGNs transfected with the gene RBMX grown on CSPG (C) or laminin (D) substrates, or the peroxidase GPX3 grown on CSPGs (E) or laminin (F). G–J, Adult brain expression of four clones with significant phenotypic changes. Data were analyzed from the Allen Brain Atlas (www.brain-map.org) to determine the expression patterns and intensities of the active genes. In-situ hybridization demonstrated little mRNA expression for GPX3 (G), and RMBX (H), each of which promotes neurite growth. EIF2B5 (I), which also promoted growth had some expression throughout the brain, especially in CA1 and the dentate gyrus. Two of the inhibitory genes, SMARCAL1 (not shown) and DUS3L (J) had strong expression in the granule layer of the cerebellum. Expression intensity legend on the far right. Scale bar in C–E 200 µm. Scale bar in G–J 1 mm.
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pone-0038101-g003: Active growth genes in cerebellar neurons and their expression pattern.A, B, Phenotypic results of overexpression in cerebellar granule neurons (CGNs) confirmed by four replicates following the primary screen. Bars represent normalized values, centered on the neutral control, mCherry. Three parameters: number of branches (gray), neurite total length (white) and primary neurite count (black), are reported for transfected neurons (GFP positive). Significant results are indicated with asterisks (ANOVA). Genes had effects on CSPG (A), laminin (B), or both substrates. mCherry transfection with Gö6976, the positive control, is plotted on the far right. C–F, Representative images of CGNs transfected with the gene RBMX grown on CSPG (C) or laminin (D) substrates, or the peroxidase GPX3 grown on CSPGs (E) or laminin (F). G–J, Adult brain expression of four clones with significant phenotypic changes. Data were analyzed from the Allen Brain Atlas (www.brain-map.org) to determine the expression patterns and intensities of the active genes. In-situ hybridization demonstrated little mRNA expression for GPX3 (G), and RMBX (H), each of which promotes neurite growth. EIF2B5 (I), which also promoted growth had some expression throughout the brain, especially in CA1 and the dentate gyrus. Two of the inhibitory genes, SMARCAL1 (not shown) and DUS3L (J) had strong expression in the granule layer of the cerebellum. Expression intensity legend on the far right. Scale bar in C–E 200 µm. Scale bar in G–J 1 mm.

Mentions: We selected a set of 79 genes from the primary screen to study in a secondary assay. Gene selection was based on ability to perturb the following parameters: neurite initiation, neurite length, neurite branching, neuron count, viability, and transfection rates, in either a positive or negative direction (including some of the genes listed in Figure 2A). The secondary screen challenged CGNs transfected with one of the genes on CSPGs and laminin in four experimental replicates. Sixteen of these genes had significant effects for a specific neurite outgrowth parameter (Table S3), a selection of which is depicted in Figure 3 compared to mCherry control in neurons plated on CSPGs (Fig. 3A), or laminin (Fig. 3B). The RNA binding motif protein, RBMX, was confirmed to increase neurite initiation on laminin (Fig. 3D) but not on CSPGs (Fig. 3C). On CSPGs, the extracellular peroxidase GPX3 allowed neurons to overcome inhibition, with little effect on laminin (Fig. 3E,F) both in transfected and non-transfected neurons. Since non-transfected neurons were affected, it is possible that GPX3’s action is extracellular. Interestingly, a translation factor implicated in neural development, EIF2B5 (translation initiation factor 2B5), also potentiated neurite growth on CSPGs. The genes SMARCAL1 (SWI/SNF related, matrix associated, actin-dependent regulator of chromatin), SDPR (serum deprivation response), DUS3L (dihydrouridine synthase, see table), and to a lesser extent ANXA2 (Annexin A2) and IGH-6 (LOC636126) inhibited growth on laminin and/or CSPGs. Interestingly, two of the inhibitors, EIF2B5 and DUS3L were highly expressed in the cerebellar granular layer of adult mice (Fig. 3I, J). Cerebellar enriched genes like these were expected to be present in the library at a rate of about three percent (Figure S1), opposed to the others, which were absent from the cerebellum and most of the CNS (Fig. 3G, H). The confirmed observation of their inhibitory nature suggests they may be targets to antagonize in order to enhance regeneration.


Peripheral nervous system genes expressed in central neurons induce growth on inhibitory substrates.

Buchser WJ, Smith RP, Pardinas JR, Haddox CL, Hutson T, Moon L, Hoffman SR, Bixby JL, Lemmon VP - PLoS ONE (2012)

Active growth genes in cerebellar neurons and their expression pattern.A, B, Phenotypic results of overexpression in cerebellar granule neurons (CGNs) confirmed by four replicates following the primary screen. Bars represent normalized values, centered on the neutral control, mCherry. Three parameters: number of branches (gray), neurite total length (white) and primary neurite count (black), are reported for transfected neurons (GFP positive). Significant results are indicated with asterisks (ANOVA). Genes had effects on CSPG (A), laminin (B), or both substrates. mCherry transfection with Gö6976, the positive control, is plotted on the far right. C–F, Representative images of CGNs transfected with the gene RBMX grown on CSPG (C) or laminin (D) substrates, or the peroxidase GPX3 grown on CSPGs (E) or laminin (F). G–J, Adult brain expression of four clones with significant phenotypic changes. Data were analyzed from the Allen Brain Atlas (www.brain-map.org) to determine the expression patterns and intensities of the active genes. In-situ hybridization demonstrated little mRNA expression for GPX3 (G), and RMBX (H), each of which promotes neurite growth. EIF2B5 (I), which also promoted growth had some expression throughout the brain, especially in CA1 and the dentate gyrus. Two of the inhibitory genes, SMARCAL1 (not shown) and DUS3L (J) had strong expression in the granule layer of the cerebellum. Expression intensity legend on the far right. Scale bar in C–E 200 µm. Scale bar in G–J 1 mm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368946&req=5

pone-0038101-g003: Active growth genes in cerebellar neurons and their expression pattern.A, B, Phenotypic results of overexpression in cerebellar granule neurons (CGNs) confirmed by four replicates following the primary screen. Bars represent normalized values, centered on the neutral control, mCherry. Three parameters: number of branches (gray), neurite total length (white) and primary neurite count (black), are reported for transfected neurons (GFP positive). Significant results are indicated with asterisks (ANOVA). Genes had effects on CSPG (A), laminin (B), or both substrates. mCherry transfection with Gö6976, the positive control, is plotted on the far right. C–F, Representative images of CGNs transfected with the gene RBMX grown on CSPG (C) or laminin (D) substrates, or the peroxidase GPX3 grown on CSPGs (E) or laminin (F). G–J, Adult brain expression of four clones with significant phenotypic changes. Data were analyzed from the Allen Brain Atlas (www.brain-map.org) to determine the expression patterns and intensities of the active genes. In-situ hybridization demonstrated little mRNA expression for GPX3 (G), and RMBX (H), each of which promotes neurite growth. EIF2B5 (I), which also promoted growth had some expression throughout the brain, especially in CA1 and the dentate gyrus. Two of the inhibitory genes, SMARCAL1 (not shown) and DUS3L (J) had strong expression in the granule layer of the cerebellum. Expression intensity legend on the far right. Scale bar in C–E 200 µm. Scale bar in G–J 1 mm.
Mentions: We selected a set of 79 genes from the primary screen to study in a secondary assay. Gene selection was based on ability to perturb the following parameters: neurite initiation, neurite length, neurite branching, neuron count, viability, and transfection rates, in either a positive or negative direction (including some of the genes listed in Figure 2A). The secondary screen challenged CGNs transfected with one of the genes on CSPGs and laminin in four experimental replicates. Sixteen of these genes had significant effects for a specific neurite outgrowth parameter (Table S3), a selection of which is depicted in Figure 3 compared to mCherry control in neurons plated on CSPGs (Fig. 3A), or laminin (Fig. 3B). The RNA binding motif protein, RBMX, was confirmed to increase neurite initiation on laminin (Fig. 3D) but not on CSPGs (Fig. 3C). On CSPGs, the extracellular peroxidase GPX3 allowed neurons to overcome inhibition, with little effect on laminin (Fig. 3E,F) both in transfected and non-transfected neurons. Since non-transfected neurons were affected, it is possible that GPX3’s action is extracellular. Interestingly, a translation factor implicated in neural development, EIF2B5 (translation initiation factor 2B5), also potentiated neurite growth on CSPGs. The genes SMARCAL1 (SWI/SNF related, matrix associated, actin-dependent regulator of chromatin), SDPR (serum deprivation response), DUS3L (dihydrouridine synthase, see table), and to a lesser extent ANXA2 (Annexin A2) and IGH-6 (LOC636126) inhibited growth on laminin and/or CSPGs. Interestingly, two of the inhibitors, EIF2B5 and DUS3L were highly expressed in the cerebellar granular layer of adult mice (Fig. 3I, J). Cerebellar enriched genes like these were expected to be present in the library at a rate of about three percent (Figure S1), opposed to the others, which were absent from the cerebellum and most of the CNS (Fig. 3G, H). The confirmed observation of their inhibitory nature suggests they may be targets to antagonize in order to enhance regeneration.

Bottom Line: Peripheral nervous system (PNS) neurons exhibit increased regenerative ability compared to central nervous system neurons, even in the presence of inhibitory environments.Several known growth associated proteins potentiated neurite growth on laminin.Bioinformatic approaches also uncovered a number of novel gene families that altered neurite growth of CNS neurons.

View Article: PubMed Central - PubMed

Affiliation: Miami Project to Cure Paralysis, Department of Pharmacology, University of Miami, Miller School of Medicine, Miami, Florida, United States of America.

ABSTRACT
Trauma to the spinal cord and brain can result in irreparable loss of function. This failure of recovery is in part due to inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans (CSPGs). Peripheral nervous system (PNS) neurons exhibit increased regenerative ability compared to central nervous system neurons, even in the presence of inhibitory environments. Previously, we identified over a thousand genes differentially expressed in PNS neurons relative to CNS neurons. These genes represent intrinsic differences that may account for the PNS's enhanced regenerative ability. Cerebellar neurons were transfected with cDNAs for each of these PNS genes to assess their ability to enhance neurite growth on inhibitory (CSPG) or permissive (laminin) substrates. Using high content analysis, we evaluated the phenotypic profile of each neuron to extract meaningful data for over 1100 genes. Several known growth associated proteins potentiated neurite growth on laminin. Most interestingly, novel genes were identified that promoted neurite growth on CSPGs (GPX3, EIF2B5, RBMX). Bioinformatic approaches also uncovered a number of novel gene families that altered neurite growth of CNS neurons.

Show MeSH
Related in: MedlinePlus