Phosphatase and tensin homologue/protein kinase B pathway linked to motor neuron survival in human superoxide dismutase 1-related amyotrophic lateral sclerosis

Abstract

Gene expression profiling has been used previously with spinal cord homogenates and laser capture microdissected motor neurons to determine the mechanisms involved in neurodegeneration in amyotrophic lateral sclerosis. However, while cellular and animal model work has focused on superoxide dismutase 1-related amyotrophic lateral sclerosis, the transcriptional profile of human mutant superoxide dismutase 1 motor neurons has remained undiscovered. The aim of this study was to apply gene expression profiling to laser captured motor neurons from human superoxide dismutase 1-related amyotrophic lateral sclerosis and neurologically normal control cases, in order to determine those pathways dysregulated in human superoxide dismutase 1-related neurodegeneration and to establish potential pathways suitable for therapeutic intervention. Identified targets were then validated in cultured cell models using lentiviral vectors to manipulate the expression of key genes. Microarray analysis identified 1170 differentially expressed genes in spinal cord motor neurons from superoxide dismutase 1-related amyotrophic lateral sclerosis, compared with controls. These genes encoded for proteins in multiple functional categories, including those involved in cell survival and cell death. Further analysis determined that multiple genes involved in the phosphatidylinositol-3 kinase signalling cascade were differentially expressed in motor neurons that survived the disease process. Functional experiments in cultured cells and primary motor neurons demonstrate that manipulating this pathway by reducing the expression of a single upstream target, the negative phosphatidylinositol-3 kinase regulator phosphatase and tensin homology, promotes a marked pro-survival effect. Therefore, these data indicate that proteins in the phosphatidylinositol-3 kinase pathway could represent a target for therapeutic manipulation in motor neuron degeneration.

Figure 1

Interacting pathways of cell survival present in the mtSOD1 motor neurons. (A) Increase in PIK3CD, which inhibits RHOA and PTEN, (B) the connection between PIP2, phospholipase CD1 (PLCphosp), diacylglycerol (DAG) and protein kinase C epsilon (PRKCEpr). (C) Increased protein kinase C epsilon activates AKT3 and increases BCL2 and inhibits Bcl2-antagonist of death expression (BAD) and (D) shows how the increase in monoacylglycerol O-transferase (MOGAT) and decrease in diacylglycerol O-acyltransferase (DGAT) ensure levels of diacylglycerol are available for protein kinase C epsilon activation. AKT3-P = phophorylated AKT3; IP3 = inositol 1,4,5 triphosphate; ITGB1be = beta-1-integrin; MAG = monoacylglycerol; PI4P = phosphatidylinositol-4 phosphate; PIK3CD = phosphatidylinositol-3 kinase catalytic subunit delta; PIP2 = phosphatidylinositol-4,5-bisphosphate; RHOA = ras homology gene family member A; SYNJ2 = synaptojanin 2; TAG = triacylglycerol.

Figure 2

Results of transduction of NSC34 cells with small interfering RNA against PTEN (siPTEN) and scrambled small interfering RNA against PTEN (SsiPTEN). (A) Representative western blot illustrating the knockdown of PTEN expression in the presence of the small interfering RNA against PTEN. The control is untransfected NSC34 cells. (B) Graph showing the level of PTEN protein expression in control and in small interfering RNA against PTEN and scrambled small interfering RNA against PTEN transduced (green fluorescent protein positive) NSC34 cells relative to GAPDH (n = 4; **P < 0.01; values represent means ± SD). (C) Effect of PTEN knockdown on survival of untransfected NSC34 cells and (D) survival of G93A SOD1 stably transfected NSC34 cells, following treatment with 10 mM H₂O₂ for 2 h (n = 4; *P < 0.05; values represent means ± SD). GFP = green fluorescent protein.

Figure 3

Western blotting of primary motor neuronal cultures transfected with constructs over expressing wild-type (WT) or C124S mutant PTEN or transduced with small interfering RNA against PTEN (siPTEN) and scrambled small interfering RNA against PTEN (SsiPTEN). Using antibodies against (A) PTEN, (B) phosphorylated AKT and (C) phosphorylated Bcl2-antagonist of death, reduced PTEN expression (A) is shown to be associated with increased phosphorylation of AKT and Bcl2-antagonist of death (B, C). In each graph: lane 1 = wild-type PTEN; lane 2 = dominant negative mutant C124S PTEN; lane 3 = untransfected primary motor neurons; lane 4 = scrambled small interfering RNA against PTEN; and lane 5 = small interfering RNA against PTEN (n = 3; *P < 0.05; values represent means ± SD).

Figure 4

Results of transduction of primary motor neuronal cultures originating from G93A SOD1 mice and litter mates with small interfering RNA (siRNA) against PTEN and scrambled small interfering RNA against PTEN (ssiPTEN). Antibodies against (A) PTEN, (C) phosphorylated AKT and (D) phosphorylated Bcl2-antagonist of death (BAD) show the concomitant decrease in expression of PTEN and increase in phosphorylation of AKT and Bcl2-antagonist of death (n = 3; *P < 0.05, **P < 0.01; values represent means ± SD). (B) Survival of motor neuron in transduced primary motor neuronal cultures on Day 7 (n = 3; *P < 0.05, **P < 0.01; values represent means ± SD). MN = motor neurons; wt = wild-type.