Modulation of LPA receptor expression in the human brain following neurotrauma

Abstract

Lysophosphatidic acid (LPA) is involved in physiological and pathological states, including in neural development and inflammation. We assessed the expression pattern of the LPA receptors 1-3 and of LPA-producing enzyme autotaxin in post-mortem human brain tissue, both in normal individuals and in individuals who died following traumatic brain injury. We found that LPA receptors and autotaxin are weakly expressed in the normal control adult brain. Quantitative PCR for the LPA receptors and autotaxin mRNA showed an increase of LPAR(2) and a decrease of autotaxin mRNA expression in the cortex following brain injury. Immunohistochemical analysis showed that LPAR(1) colocalized with astrocytes and that LPAR(2) is present on the ependymal cells lining the lateral ventricle in the brain samples from individuals who died following severe head injury. This work shows for the first time that key components of the LPA pathway are modulated following TBI in humans.

Fig. 1

Validation of endogenous control genes for Q-PCR in human post mortem brain tissue. a Average expression stability values M show that HMBS, PPIA, UBC and GAPDH are the endogenous control genes with the most stable mRNA levels while B2M, SDHA and TBP are the gene with the least stable mRNA levels. b Pairwise variations indicate that a minimum of four endogenous control genes is needed (V < 0.15) for normalisation for Q-PCR data in post mortem human brain tissue

Fig. 2

LPAR and ATX mRNA levels in post mortem human cortex following TBI. Q-PCR for ATX (a) and LPAR_1-3 (b–d) in control, acute, early and late groups. Statistical significance: *: P = 0.013 (a), P = 0.047 (c), one-way ANOVA. Arbitrary unit; mRNA control levels = 1

Fig. 3

Demonstration of LPAR antibody specificities in HepG2 cells. a Q-PCR for LPARs and ATX. b Q-PCR analysis of LPAR₂ mRNA expression at 24, 48, 72 h following transfection with human LPAR₂ siRNA. Data are the mean ± SEM of triplicate samples of 2^−∆∆ct with data normalized to expression levels of the housekeeping genes HPRT, β-actin and GAPDH and relative to LPAR₂. Statistical significance: *P < 0.05, **P < 0.01 and ***P < 0.001 by one-tailed t-test. c Immunostaining with GFP (green) and LPAR₁ (red) of cells transfected or not (Control) with human LPAR₁ clone (Overexpression), with DAPI counterstain at two different magnifications. d Immunostaining with LPAR₂ of cells transfected (siRNA) or not with human LPAR₂ small interference RNA Smartpool, with DAPI counterstain. Scale bars 100 μm (Color figure online)

Fig. 4

Modulation of LPAR expression following TBI. Expression of the LPAR_1-3 was assessed in non-injured (Normal, a) and injured brain tissues (corpus callosum (CC) and cerebral cortex) at various time of trauma (Late, b–d, f–h). Reactive astrocytes in late tissues were assessed by GFAP (e). i Negative controls. Scale bar 100 μm. LV: lateral ventricle. Arrows indicate representative staining