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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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Cerebellar granule neurons are robustly inhibited by CSPGs and partially rescued by Gö6976.Dissociated postnatal cerebellar granule neurons (CGNs) were challenged in an inhibitory assay. A, B, representative images of CGNs transfected with control plasmid pSport mCherry growing on CSPGs (A), and a permissive substrate, laminin (B). C,D, the addition of the PKC inhibitor Gö6976 partially relieved CSPG inhibition (C), and potentiated laminin growth (D). E, Bar chart depicting ratios of seven parameters on CSPGs divided by laminin (with 95% confidence intervals). Growth on CSPGs led to large decreases in neurite length, the number of primary neurites, and the percentage of cells with neurites. Tubulin intensity and soma area were slightly increased. The neuron count was decreased on CSPGs, implying a deficiency in cell adhesion or survival. F, Percent increase by Gö6976 was plotted with 95% confidence intervals on CSPGs (open bars), and on laminin (solid bars). Gö6976 had a strong positive effect on neurons growing on CSPGs, especially for neurite length and the percentage of neurons growing neurites. Neuron count and viability was unchanged. Statistics with un-normalized treatment averages, Mann-Whitney Test, *p< = 0.05, **p< = 0.01, ***p< = 0.0001. Scale bar 100 µm.
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pone-0038101-g001: Cerebellar granule neurons are robustly inhibited by CSPGs and partially rescued by Gö6976.Dissociated postnatal cerebellar granule neurons (CGNs) were challenged in an inhibitory assay. A, B, representative images of CGNs transfected with control plasmid pSport mCherry growing on CSPGs (A), and a permissive substrate, laminin (B). C,D, the addition of the PKC inhibitor Gö6976 partially relieved CSPG inhibition (C), and potentiated laminin growth (D). E, Bar chart depicting ratios of seven parameters on CSPGs divided by laminin (with 95% confidence intervals). Growth on CSPGs led to large decreases in neurite length, the number of primary neurites, and the percentage of cells with neurites. Tubulin intensity and soma area were slightly increased. The neuron count was decreased on CSPGs, implying a deficiency in cell adhesion or survival. F, Percent increase by Gö6976 was plotted with 95% confidence intervals on CSPGs (open bars), and on laminin (solid bars). Gö6976 had a strong positive effect on neurons growing on CSPGs, especially for neurite length and the percentage of neurons growing neurites. Neuron count and viability was unchanged. Statistics with un-normalized treatment averages, Mann-Whitney Test, *p< = 0.05, **p< = 0.01, ***p< = 0.0001. Scale bar 100 µm.

Mentions: We hypothesized that DRG neurons selectively express regeneration-associated genes, which if overexpressed in CNS neurons could promote axon regeneration. We therefore tested the neurite growth promoting ability of each PNS gene in cerebellar granule neurons (CGNs). CGNs are widely used in the study of neurite growth inhibition [25], [26], [27], and to this end we developed an inhibitory growth assay. We challenged postnatal mouse CGNs to grow on an inhibitory substrate, comparing their neurite extension with control neurons grown on permissive substrates. We initially developed a neurite outgrowth assay using CNS myelin as an inhibitory substrate, which was used in a small preliminary screen (Table S1). Because this proved difficult to scale up for a full screen we used a mixture of chondroitin sulfate proteoglycans and laminin (referred to as simply CSPGs) as the inhibitory substrate and laminin alone as the permissive substrate [28]. Postnatal CGNs exhibited a normal bipolar morphology on laminin (Fig. 1B) and were strongly inhibited by CSPGs (Fig. 1A). Principle component analysis and strictly standardized mean difference (SSMD) analysis indicated that total neurite count and length were the most reliable feature relating to neurite growth of the dozen features reported by the Cellomics Neuronal Profiling Bio-application. The total neurite length for each neuron was reduced fivefold on CSPGs (p<10−29, Mann-Whitney U test [MWU]). The number of primary neurites and the percent of neurons that initiated neurite growth were each reduced about threefold on CSPGs compared to laminin (Fig. 1E). Growth inhibition by CSPGs was present at all basal levels of neurite outgrowth on laminin, which varied among experiments (Figure S3). Intensity of tubulin staining and the area of the soma were slightly but significantly increased on CSPGs (5% and 9% respectively, Fig. 1E). Dead neurons were easily distinguished by positive Hoechst signal with no accompanying tubulin positive soma. This parameter (the ratio of neurons per nucleus) is a rough measure of neuronal viability, and was increased slightly in CSPGs compared to laminin (Fig. 1E). Thus, CSPGs robustly inhibited neurite growth, reduced adhesion, and didn’t have a strong effect on viability.


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)

Cerebellar granule neurons are robustly inhibited by CSPGs and partially rescued by Gö6976.Dissociated postnatal cerebellar granule neurons (CGNs) were challenged in an inhibitory assay. A, B, representative images of CGNs transfected with control plasmid pSport mCherry growing on CSPGs (A), and a permissive substrate, laminin (B). C,D, the addition of the PKC inhibitor Gö6976 partially relieved CSPG inhibition (C), and potentiated laminin growth (D). E, Bar chart depicting ratios of seven parameters on CSPGs divided by laminin (with 95% confidence intervals). Growth on CSPGs led to large decreases in neurite length, the number of primary neurites, and the percentage of cells with neurites. Tubulin intensity and soma area were slightly increased. The neuron count was decreased on CSPGs, implying a deficiency in cell adhesion or survival. F, Percent increase by Gö6976 was plotted with 95% confidence intervals on CSPGs (open bars), and on laminin (solid bars). Gö6976 had a strong positive effect on neurons growing on CSPGs, especially for neurite length and the percentage of neurons growing neurites. Neuron count and viability was unchanged. Statistics with un-normalized treatment averages, Mann-Whitney Test, *p< = 0.05, **p< = 0.01, ***p< = 0.0001. Scale bar 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038101-g001: Cerebellar granule neurons are robustly inhibited by CSPGs and partially rescued by Gö6976.Dissociated postnatal cerebellar granule neurons (CGNs) were challenged in an inhibitory assay. A, B, representative images of CGNs transfected with control plasmid pSport mCherry growing on CSPGs (A), and a permissive substrate, laminin (B). C,D, the addition of the PKC inhibitor Gö6976 partially relieved CSPG inhibition (C), and potentiated laminin growth (D). E, Bar chart depicting ratios of seven parameters on CSPGs divided by laminin (with 95% confidence intervals). Growth on CSPGs led to large decreases in neurite length, the number of primary neurites, and the percentage of cells with neurites. Tubulin intensity and soma area were slightly increased. The neuron count was decreased on CSPGs, implying a deficiency in cell adhesion or survival. F, Percent increase by Gö6976 was plotted with 95% confidence intervals on CSPGs (open bars), and on laminin (solid bars). Gö6976 had a strong positive effect on neurons growing on CSPGs, especially for neurite length and the percentage of neurons growing neurites. Neuron count and viability was unchanged. Statistics with un-normalized treatment averages, Mann-Whitney Test, *p< = 0.05, **p< = 0.01, ***p< = 0.0001. Scale bar 100 µm.
Mentions: We hypothesized that DRG neurons selectively express regeneration-associated genes, which if overexpressed in CNS neurons could promote axon regeneration. We therefore tested the neurite growth promoting ability of each PNS gene in cerebellar granule neurons (CGNs). CGNs are widely used in the study of neurite growth inhibition [25], [26], [27], and to this end we developed an inhibitory growth assay. We challenged postnatal mouse CGNs to grow on an inhibitory substrate, comparing their neurite extension with control neurons grown on permissive substrates. We initially developed a neurite outgrowth assay using CNS myelin as an inhibitory substrate, which was used in a small preliminary screen (Table S1). Because this proved difficult to scale up for a full screen we used a mixture of chondroitin sulfate proteoglycans and laminin (referred to as simply CSPGs) as the inhibitory substrate and laminin alone as the permissive substrate [28]. Postnatal CGNs exhibited a normal bipolar morphology on laminin (Fig. 1B) and were strongly inhibited by CSPGs (Fig. 1A). Principle component analysis and strictly standardized mean difference (SSMD) analysis indicated that total neurite count and length were the most reliable feature relating to neurite growth of the dozen features reported by the Cellomics Neuronal Profiling Bio-application. The total neurite length for each neuron was reduced fivefold on CSPGs (p<10−29, Mann-Whitney U test [MWU]). The number of primary neurites and the percent of neurons that initiated neurite growth were each reduced about threefold on CSPGs compared to laminin (Fig. 1E). Growth inhibition by CSPGs was present at all basal levels of neurite outgrowth on laminin, which varied among experiments (Figure S3). Intensity of tubulin staining and the area of the soma were slightly but significantly increased on CSPGs (5% and 9% respectively, Fig. 1E). Dead neurons were easily distinguished by positive Hoechst signal with no accompanying tubulin positive soma. This parameter (the ratio of neurons per nucleus) is a rough measure of neuronal viability, and was increased slightly in CSPGs compared to laminin (Fig. 1E). Thus, CSPGs robustly inhibited neurite growth, reduced adhesion, and didn’t have a strong effect on viability.

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