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. 2015 Jun;37(3):62.
doi: 10.1007/s11357-015-9789-6. Epub 2015 Jun 6.

Analysis of gene expression during aging of CGNs in culture: implication of SLIT2 and NPY in senescence

K Preeti Gupta  1 , Pankaj Singh Dholaniya, Anil Chekuri, Anand K Kondapi
Affiliations

Analysis of gene expression during aging of CGNs in culture: implication of SLIT2 and NPY in senescence

K Preeti Gupta et al. Age (Dordr). 2015 Jun.

Erratum in

Abstract

Senescence is the major key factor that leads to the loss of neurons throughout aging. Cellular senescence is not the consequence of single cause, but there are multiple aspects which may induce senescence in a cell. Various causes such as gene expression, molecular interactions and protein processing and chromatin organization are described as causal factor for senescence. It is well known that the damage to the nuclear or mitochondrial DNA contributes to the aging either directly by inducing the apoptosis/cellular senescence or indirectly by altering cellular functions. The significant nuclear DNA damage with the age is directly associated with the continuous declining in DNA repair. The continuous decline in expression of topoisomerase 2 beta (Topo IIβ) in cultured cerebellar granule neurons over time indicated the decline in the repair of damage DNA. DNA Topo IIβ is an enzyme that is crucial for solving topological problems of DNA and thus has an important role in DNA repair. The enzyme is predominantly present in non-proliferating cells such as neurons. In this paper, we have studied the genes which were differentially expressed over time in cultured cerebellar granule neurons (CGNs) and identified potential genes associated with the senescence. Our results showed that the two genes neuropeptide Y (Npy) and Slit homolog 2 (Drosophila) (Slit2) gradually increase during aging, and upon suppression of these two genes, there was gradual increase in cell viability along with restoration of the expression of Topo IIβ and potential repair proteins.

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Figures

Graphical abstract
Graphical abstract
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Fig. 1
Fig. 1
Heat map of differentially expressed genes in ageing CGNs. A heat map was created for the entire genome to display those statistically significant (P value <0.05 with multiple testing corrections applied). Fold change used for upregulation and downregulation was 0.8 in each of the replicates and >1 in the Geomean fold of the replicated samples. Normalized expression signals are represented on a log scale for which colder colors correspond to lower levels of expression and warmer colors correspond to higher levels of expression. Genes were clustered, and heat maps were generated using Cluster v3.0/TreeView (de Hoon et al. ; Eisen et al. 1998). Panels a–c show comparisons of other weeks with 1, 2, and 3 W as in set 1, set 2, and set 3, respectively
Fig. 2
Fig. 2
Heat map of selected differentially upregulated genes. A heat map was created to display those statistically significant genes (P value <0.05 with multiple testing corrections applied) upregulated genes having fold change more than 1.55 in second week. Normalized expression signals are represented on a log scale for which colder colors correspond to lower levels of expression and warmer colors correspond to higher levels of expression. Genes were clustered and heat maps were generated using Cluster v3.0/TreeView (de Hoon et al. ; Eisen et al. 1998)
Fig. 3
Fig. 3
Gene Ontology Classification of differentially expressed genes based on function. Gene Ontology of microarray differentials representing number of genes downregulated and upregulated in ageing CGNs from 1 to 4 W is shown in the bar chart. Fold change value of 0.8 and P value <0.05 was considered to be statistically significant. The GO classification was performed by “PANTHER Classification System” (http://www.pantherdb.org/)
Fig. 4
Fig. 4
Validation of differential gene expression by qPCR. Panels a and b show fold change expression by microarray analysis. Panels c and d depict validation by q-RT PCR of genes—A2m, Gna14, Gria1, Masp1, Npy, Slit2, and Glb1. q-RT PCR analysis reveals significant increase in relative expression of all genes through the weeks as per statistical analysis, one-way ANOVA (Tukey’s post hoc test) (*P < 0.05, **P < 0.01, ***P < 0.001). 1 W healthy CGNs were taken as control. Values are represented as mean ± SD and n = 6. Comparative CT method was employed to evaluate the gene transcript pattern in ageing cultures
Fig. 5
Fig. 5
Semi quantitative PCR analysis. Panels a and b show semi-quantitative gene expression of A2m, Gna14, Gria1, Masp1, Npy, Slit2, and Glb1. All the genes showed significant increased expression in 2, 3, and 4 W over 1 W. Lanes 1–4 correspond to 1 to 4 W transcript levels, lane 5 is no template control (NTC), and lane 6 is 50 bp low range ladder
Fig. 6
Fig. 6
Figure shows cytotoxicity detected by reduction of MTT in ageing CGNs. Results are expressed as mean ± SD (three replicates in two independent experiments). Cell viability in first week was considered as 100 %. Values are presented as a percentage of activity in control cells. Cell viability is compared at every week based on MTT values presented. The results showed a marked increase in cell viability in Npy and Slit2 downregulated cells compared to control, when knockdown was done at third week in culture
Fig. 7
Fig. 7
Expression of Topo IIβ in Slit2 and Npy downregulated cells. Relative mRNA levels of Topo IIβ in downregulated cells were determined by real-time PCR. Genes show a significant increase in relative gene expression through the weeks as per statistical analysis; 1 W healthy CGNs were taken as control
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References

    1. Baker DJ, et al. Opposing roles for p16Ink4a and p19Arf in senescence and ageing caused by BubR1 insufficiency. Nat Cell Biol. 2008;10:825–836. doi: 10.1038/ncb1744. - DOI - PMC - PubMed
    1. Beausejour CM, Krtolica A, Galimi F, Narita M, Lowe SW, Yaswen P, Campisi J. Reversal of human cellular senescence: roles of the p53 and p16 pathways. EMBO J. 2003;22:4212–4222. doi: 10.1093/emboj/cdg417. - DOI - PMC - PubMed
    1. Bhanu MU, Mandraju RK, Bhaskar C, Kondapi AK. Cultured cerebellar granule neurons as an in vitro aging model: topoisomerase IIbeta as an additional biomarker in DNA repair and aging. Toxicol in Vitro Int J Publ Assoc BIBRA. 2010;24:1935–1945. doi: 10.1016/j.tiv.2010.08.003. - DOI - PubMed
    1. Blalock EM, Porter NM, Landfield PW. Decreased G-protein-mediated regulation and shift in calcium channel types with age in hippocampal cultures. J Neurosci Off J Soc Neurosci. 1999;19:8674–8684. - PMC - PubMed
    1. Borbely S, et al. Modification of ionotropic glutamate receptor-mediated processes in the rat hippocampus following repeated, brief seizures. Neuroscience. 2009;159:358–368. doi: 10.1016/j.neuroscience.2008.12.027. - DOI - PubMed

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