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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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Identifying Gene Ontology Clusters that Regulate Axon Outgrowth.Tiled cluster analysis from Fig 6 run for Gene Ontology “Molecular Function” annotations on 675 genes. A, Cluster heat map for the parameter neurite average length on CSPGs. 7 Tiers shown, with clusters per tier from 42 (bottom, largest clusters with most divergent genes) to 479 clusters per tier (top, smallest clusters with most closely related genes). B, Region of magnification. C, Dendrogram of 96 genes for region from (B). Four ontologies define the large classes of genes in this region (although hundreds of ontologies are present). D, Cluster heat map magnified from (B). Individual gene clusters are defined by tiles where extent of change is color coded (white  =  control, red  =  reduction, green  =  positive). Legend in lower left corner. Single black square p<0.05, double p<0.01, (uncorrected bootstrap). E, Summary table of significant gene clusters from analysis of neurite average length, branching, primary neurite count, and an absolute analysis of neurite average length (see methods). Outlines around tiles indicate higher significance.
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pone-0038101-g006: Identifying Gene Ontology Clusters that Regulate Axon Outgrowth.Tiled cluster analysis from Fig 6 run for Gene Ontology “Molecular Function” annotations on 675 genes. A, Cluster heat map for the parameter neurite average length on CSPGs. 7 Tiers shown, with clusters per tier from 42 (bottom, largest clusters with most divergent genes) to 479 clusters per tier (top, smallest clusters with most closely related genes). B, Region of magnification. C, Dendrogram of 96 genes for region from (B). Four ontologies define the large classes of genes in this region (although hundreds of ontologies are present). D, Cluster heat map magnified from (B). Individual gene clusters are defined by tiles where extent of change is color coded (white  =  control, red  =  reduction, green  =  positive). Legend in lower left corner. Single black square p<0.05, double p<0.01, (uncorrected bootstrap). E, Summary table of significant gene clusters from analysis of neurite average length, branching, primary neurite count, and an absolute analysis of neurite average length (see methods). Outlines around tiles indicate higher significance.

Mentions: Our primary screen resulted in quantitative functional data for a wide range of parameters. We next asked the question “Do groups of related genes, when considered together, produce significant changes in neuronal morphology?” We assume that further meaning emerges when these genes are studied as they are in reality–in a system. To artificially reconstitute the “system”, we sought to interrogate clusters using the existing functional results from the primary screen. This method [30] is the reverse of a common practice that determines representation of ontologies in a gene list compared to background [31]. We used the “molecular function” ontology information to generate a hierarchical cluster of genes. This analysis revealed that genes within some ontological clusters had directionally consistent effects on neurite outgrowth (e.g., RPS/RPL genes tended to promote axon growth; Fig. S4). Figure 6 demonstrates the results with neurite average length for neurons growing on CSPGs (Fig. 6A). A region of the ontology space (Fig. 6B), which contains transcription factors, zinc and DNA binding proteins, ion channels, and ubiquitin ligases is shown in greater detail (Fig. 6C, D). This cluster heatmap shows individual genes affecting neurite length (top tier) by color. Further down the tier, genes grouped by molecular function (i.e. transcription factor) can be seen to affect or not affect neurite length on CSPGs. Neurite length was effectively inhibited by a small group of potassium gated channels, as well as two ubiquitin ligases (Fig. 6D). It is important to note that the estimated false discovery rate for the overall screen, based on # of primary neurites, was 24% (Methods S1) and therefore this particular analysis is likely to contain some artifacts. Most of the noise was due to variations in experimental sets from different mice and different days. Higher numbers of controls on each plate would likely reduce FDR [32].


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)

Identifying Gene Ontology Clusters that Regulate Axon Outgrowth.Tiled cluster analysis from Fig 6 run for Gene Ontology “Molecular Function” annotations on 675 genes. A, Cluster heat map for the parameter neurite average length on CSPGs. 7 Tiers shown, with clusters per tier from 42 (bottom, largest clusters with most divergent genes) to 479 clusters per tier (top, smallest clusters with most closely related genes). B, Region of magnification. C, Dendrogram of 96 genes for region from (B). Four ontologies define the large classes of genes in this region (although hundreds of ontologies are present). D, Cluster heat map magnified from (B). Individual gene clusters are defined by tiles where extent of change is color coded (white  =  control, red  =  reduction, green  =  positive). Legend in lower left corner. Single black square p<0.05, double p<0.01, (uncorrected bootstrap). E, Summary table of significant gene clusters from analysis of neurite average length, branching, primary neurite count, and an absolute analysis of neurite average length (see methods). Outlines around tiles indicate higher significance.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368946&req=5

pone-0038101-g006: Identifying Gene Ontology Clusters that Regulate Axon Outgrowth.Tiled cluster analysis from Fig 6 run for Gene Ontology “Molecular Function” annotations on 675 genes. A, Cluster heat map for the parameter neurite average length on CSPGs. 7 Tiers shown, with clusters per tier from 42 (bottom, largest clusters with most divergent genes) to 479 clusters per tier (top, smallest clusters with most closely related genes). B, Region of magnification. C, Dendrogram of 96 genes for region from (B). Four ontologies define the large classes of genes in this region (although hundreds of ontologies are present). D, Cluster heat map magnified from (B). Individual gene clusters are defined by tiles where extent of change is color coded (white  =  control, red  =  reduction, green  =  positive). Legend in lower left corner. Single black square p<0.05, double p<0.01, (uncorrected bootstrap). E, Summary table of significant gene clusters from analysis of neurite average length, branching, primary neurite count, and an absolute analysis of neurite average length (see methods). Outlines around tiles indicate higher significance.
Mentions: Our primary screen resulted in quantitative functional data for a wide range of parameters. We next asked the question “Do groups of related genes, when considered together, produce significant changes in neuronal morphology?” We assume that further meaning emerges when these genes are studied as they are in reality–in a system. To artificially reconstitute the “system”, we sought to interrogate clusters using the existing functional results from the primary screen. This method [30] is the reverse of a common practice that determines representation of ontologies in a gene list compared to background [31]. We used the “molecular function” ontology information to generate a hierarchical cluster of genes. This analysis revealed that genes within some ontological clusters had directionally consistent effects on neurite outgrowth (e.g., RPS/RPL genes tended to promote axon growth; Fig. S4). Figure 6 demonstrates the results with neurite average length for neurons growing on CSPGs (Fig. 6A). A region of the ontology space (Fig. 6B), which contains transcription factors, zinc and DNA binding proteins, ion channels, and ubiquitin ligases is shown in greater detail (Fig. 6C, D). This cluster heatmap shows individual genes affecting neurite length (top tier) by color. Further down the tier, genes grouped by molecular function (i.e. transcription factor) can be seen to affect or not affect neurite length on CSPGs. Neurite length was effectively inhibited by a small group of potassium gated channels, as well as two ubiquitin ligases (Fig. 6D). It is important to note that the estimated false discovery rate for the overall screen, based on # of primary neurites, was 24% (Methods S1) and therefore this particular analysis is likely to contain some artifacts. Most of the noise was due to variations in experimental sets from different mice and different days. Higher numbers of controls on each plate would likely reduce FDR [32].

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