The neuronal ceroid lipofuscinosis Cln8 gene expression is developmentally regulated in mouse brain and up-regulated in the hippocampal kindling model of epilepsy

Abstract

Background: The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders characterized by accumulation of autofluorescent material in many tissues, especially in neurons. Mutations in the CLN8 gene, encoding an endoplasmic reticulum (ER) transmembrane protein of unknown function, underlie NCL phenotypes in humans and mice. The human phenotype is characterized by epilepsy, progressive psychomotor deterioration and visual loss, while motor neuron degeneration (mnd) mice with a Cln8 mutation show progressive motor neuron dysfunction and retinal degeneration.

Results: We investigated spatial and temporal expression of Cln8 messenger ribonucleic acid (mRNA) using in situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR) and northern blotting. Cln8 is ubiquitously expressed at low levels in embryonic and adult tissues. In prenatal embryos Cln8 is most prominently expressed in the developing gastrointestinal tract, dorsal root ganglia (DRG) and brain. In postnatal brain the highest expression is in the cortex and hippocampus. Expression of Cln8 mRNA in the central nervous system (CNS) was also analyzed in the hippocampal electrical kindling model of epilepsy, in which Cln8 expression was rapidly up-regulated in hippocampal pyramidal and granular neurons.

Conclusion: Expression of Cln8 in the developing and mature brain suggests roles for Cln8 in maturation, differentiation and supporting the survival of different neuronal populations. The relevance of Cln8 up-regulation in hippocampal neurons of kindled mice should be further explored.

Figure 1

Northern blot and RT-PCR analysis of Cln8 mRNA expression in mouse tissues A: Northern blot analysis of Cln8 transcripts in mouse tissues. A β-actin probe was used as a control. Molecular weight marker in kb is shown on the left. B: Real-time quantitative RT-PCR analysis of Cln8 in mouse tissues. The expression of Cln8 in brain, given a value of 1, was used as a control. The expression of Cln8 in the other tissues is shown as a fold of the control.

Figure 2

In situ hybridization analysis of Cln8 mRNA expression in mouse embryos. In the E17 mouse embryo, developing gastrointestinal tract (A, B) and DRG (C, D), hybridized with Cln8 specific cRNA probe, are shown. Bright-field view of the cells is shown on the left (A, C) and dark-field detection of hybridization signals on the right (B,D). Optical density values of hybridization signals of Cln8 sense and antisense probes in E13 and E17 gut and brain tissues are shown in the diagram (E). Error bars represent standard error of the mean. s = sense, as = antisense

Figure 3

Differential distribution of Cln8 mRNA in brains of P0, P5, P10 and adult mouse. Dark-field emulsion autoradiographs from frontal sections, shown on the left, were analyzed by in situ hybridization analysis (A-D). Higher magnification in the hippocampal area CA3 is shown on the right. Bright-field views of the cells (a-d) and dark-field emulsion autoradiographs (a'-d') are shown. Optical density values of hybridization signals of Cln8 antisense probe are shown as bars in the diagram below (A = adult). Optical density values of hybridization signals of Cln8 sense probe are shown as a gray area behind the bars. In each brain region the optical density values at P0, P5 and P10 were compared to the optical density value of adult. Error bars represent standard error of the mean. Symbols *, ** and *** represent p < 0.05, 0.01 and 0.001, respectively. CX = cortex, DG = dentate gyrus

Figure 4

Distribution of Cln8 mRNA in brains of hippocampal kindling induced epileptic mice. Frontal mouse brain sections were analyzed 2 h, 6 h and 24 h after kindling induced epileptic seizures by in situ hybridization. Mice with electrodes implanted but without electrical stimulations were used as controls (0 h). Dark-field emulsion autoradiographs of hippocampal Cln8 expression in kindling-induced mice 0 h and 24 h after kindling are shown. Optical density values of hybridization signals of Cln8 antisense probe are shown as bars in the diagram. Optical density values of hybridization signals of Cln8 sense probe are shown as a gray area behind the bars. In each brain region the optical density values 2 h, 6 h and 24 h after kidling-induced seizures were compared to controls (0 h). Error bars represent standard error of the mean. Symbols *, ** and *** represent p < 0.05, 0.01 and 0.001, respectively. DG = dentate gyrus