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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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Hippocampal neurons are inhibited by CSPGs, and rescued by GPX3 overexpression.Hippocampal neurons transfected with active genes were plated on CSPG or laminin substrates. A, CSPGs (white) strongly inhibited hippocampal growth compared to laminin (black) (p<0.0001, Mann Whitney U Test) in three independent experiments (triangle, square, and circle markers). Horizontal bar indicates the average neurite total length on CSPGs and laminin, 15.5 µm and 60.9 µm respectively. B,C Mean Z-Scores of transfected neurons with standard deviations, centered on the negative control, mCherry. Asterisks indicate significant effects (*, **, ***, p<0.05, 0.01, 0.001 Tukey-Kramer) compared to the mCherry control when analyzed with ANOVA (p<0.001). D, E, Representative images of hippocampal neurons growing on CSPGs transfected with GPX3 (D) or control mCherry (E). Scale bar 100 µm.
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pone-0038101-g004: Hippocampal neurons are inhibited by CSPGs, and rescued by GPX3 overexpression.Hippocampal neurons transfected with active genes were plated on CSPG or laminin substrates. A, CSPGs (white) strongly inhibited hippocampal growth compared to laminin (black) (p<0.0001, Mann Whitney U Test) in three independent experiments (triangle, square, and circle markers). Horizontal bar indicates the average neurite total length on CSPGs and laminin, 15.5 µm and 60.9 µm respectively. B,C Mean Z-Scores of transfected neurons with standard deviations, centered on the negative control, mCherry. Asterisks indicate significant effects (*, **, ***, p<0.05, 0.01, 0.001 Tukey-Kramer) compared to the mCherry control when analyzed with ANOVA (p<0.001). D, E, Representative images of hippocampal neurons growing on CSPGs transfected with GPX3 (D) or control mCherry (E). Scale bar 100 µm.

Mentions: To extend the results of the screen to another CNS population, primary embryonic hippocampal neurons were transfected with several genes of interest in a CSPG neurite outgrowth assay. First, CSPGs strongly inhibited hippocampal neurite growth (Fig. 4A, four fold decrease, p<0.0001 MWU). The positive control used for cerebellar neurons, Gö6976, was unable to relieve CSPG inhibition for hippocampal neurons (data not shown). Interestingly, GPX3 significantly increased growth of transfected and non-transfected neurons both on CSPG (KW p = 0.019, Dunn’s post p<0.05, Fig. 4B) and on laminin (KW p<0.022 Dunn’s post p<0.05, Fig. 4C). Neurite outgrowth was also enhanced after transfection with OGFRL1, EEF2K, IVNS1ABP, and 2810452K22RIK. GPX3 significantly increased the number of neurites, length, and frequency of initiation when overexpressed in hippocampal neurons (Fig. 4D).


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)

Hippocampal neurons are inhibited by CSPGs, and rescued by GPX3 overexpression.Hippocampal neurons transfected with active genes were plated on CSPG or laminin substrates. A, CSPGs (white) strongly inhibited hippocampal growth compared to laminin (black) (p<0.0001, Mann Whitney U Test) in three independent experiments (triangle, square, and circle markers). Horizontal bar indicates the average neurite total length on CSPGs and laminin, 15.5 µm and 60.9 µm respectively. B,C Mean Z-Scores of transfected neurons with standard deviations, centered on the negative control, mCherry. Asterisks indicate significant effects (*, **, ***, p<0.05, 0.01, 0.001 Tukey-Kramer) compared to the mCherry control when analyzed with ANOVA (p<0.001). D, E, Representative images of hippocampal neurons growing on CSPGs transfected with GPX3 (D) or control mCherry (E). Scale bar 100 µm.
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Related In: Results  -  Collection

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

pone-0038101-g004: Hippocampal neurons are inhibited by CSPGs, and rescued by GPX3 overexpression.Hippocampal neurons transfected with active genes were plated on CSPG or laminin substrates. A, CSPGs (white) strongly inhibited hippocampal growth compared to laminin (black) (p<0.0001, Mann Whitney U Test) in three independent experiments (triangle, square, and circle markers). Horizontal bar indicates the average neurite total length on CSPGs and laminin, 15.5 µm and 60.9 µm respectively. B,C Mean Z-Scores of transfected neurons with standard deviations, centered on the negative control, mCherry. Asterisks indicate significant effects (*, **, ***, p<0.05, 0.01, 0.001 Tukey-Kramer) compared to the mCherry control when analyzed with ANOVA (p<0.001). D, E, Representative images of hippocampal neurons growing on CSPGs transfected with GPX3 (D) or control mCherry (E). Scale bar 100 µm.
Mentions: To extend the results of the screen to another CNS population, primary embryonic hippocampal neurons were transfected with several genes of interest in a CSPG neurite outgrowth assay. First, CSPGs strongly inhibited hippocampal neurite growth (Fig. 4A, four fold decrease, p<0.0001 MWU). The positive control used for cerebellar neurons, Gö6976, was unable to relieve CSPG inhibition for hippocampal neurons (data not shown). Interestingly, GPX3 significantly increased growth of transfected and non-transfected neurons both on CSPG (KW p = 0.019, Dunn’s post p<0.05, Fig. 4B) and on laminin (KW p<0.022 Dunn’s post p<0.05, Fig. 4C). Neurite outgrowth was also enhanced after transfection with OGFRL1, EEF2K, IVNS1ABP, and 2810452K22RIK. GPX3 significantly increased the number of neurites, length, and frequency of initiation when overexpressed in hippocampal neurons (Fig. 4D).

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