Brain gene expression profiles of Cln1 and Cln5 deficient mice unravels common molecular pathways underlying neuronal degeneration in NCL diseases

Abstract

Background: The neuronal ceroid lipofuscinoses (NCL) are a group of children's inherited neurodegenerative disorders, characterized by blindness, early dementia and pronounced cortical atrophy. The similar pathological and clinical profiles of the different forms of NCL suggest that common disease mechanisms may be involved. To explore the NCL-associated disease pathology and molecular pathways, we have previously produced targeted knock-out mice for Cln1 and Cln5. Both mouse-models replicate the NCL phenotype and neuropathology; the Cln1-/- model presents with early onset, severe neurodegenerative disease, whereas the Cln5-/- model produces a milder disease with a later onset.

Results: Here we have performed quantitative gene expression profiling of the cortex from 1 and 4 month old Cln1-/- and Cln5-/- mice. Combined microarray datasets from both mouse models exposed a common affected pathway: genes regulating neuronal growth cone stabilization display similar aberrations in both models. We analyzed locus specific gene expression and showed regional clustering of Cln1 and three major genes of this pathway, further supporting a close functional relationship between the corresponding gene products; adenylate cyclase-associated protein 1 (Cap1), protein tyrosine phosphatase receptor type F (Ptprf) and protein tyrosine phosphatase 4a2 (Ptp4a2). The evidence from the gene expression data, indicating changes in the growth cone assembly, was substantiated by the immunofluorescence staining patterns of Cln1-/- and Cln5-/- cortical neurons. These primary neurons displayed abnormalities in cytoskeleton-associated proteins actin and beta-tubulin as well as abnormal intracellular distribution of growth cone associated proteins GAP-43, synapsin and Rab3.

Conclusion: Our data provide the first evidence for a common molecular pathogenesis behind neuronal degeneration in INCL and vLINCL. Since CLN1 and CLN5 code for proteins with distinct functional roles these data may have implications for other forms of NCLs as well.

Figure 1

Common affected pathways in Cln1 and Cln5 deficient mouse models. A schematic picture of protein-protein interactions between genes that are differentially expressed in both mouse-models. Upregulated genes are shown in red and downregulated genes in green. Genes with no changes in expression are shown in blue. Genes with changes only in one replicate experiment are shown in pink.

Figure 2

Gene expression differences according to gene ontology classes in: Cln1-/- 1mo array, Cln1-/- 4mo array, Cln5-/- 1mo array, Cln5-/- 4mo array. All the significant categories are shown.

Figure 3

Chromosomal location of coregulated genes. Several differentially expressed genes show significant clustering in around Ppt1 on mouse chromosome 4, and might therefore be coregulated. The probesets for Ppt1 were excluded from the dataset and thus Ppt1 is shown in gray. The genes participating in the regulated loci or being independently regulated are shown, together with their location in base pairs in mouse chromosome 4. Genes that exhibit expression differences in both mouse models are indicated with *. Expression changes only in the Cln1-/- mouse are indicated with **.

Figure 4

A) Immunofluorescence analysis of actin and β-tubulin staining in E15 primary cortical neurons of wt, Cln1-/- and Cln5-/- mice. Actin is visualized in green and β-tubulin in red. The overlay shows the colocalization of the two proteins in yellow. B) Quantitative threshold imaging of actin and β-tubulin staining in the Cln5-/- mouse brain. Significant p-values, with p < 0.05 are indicated with *. Ctx, cortex and cc, corpus callosum.

Figure 5

A) Immunofluorescence analysis of Synapsin, Rab3 and GAP-43 staining in E15 primary cortical neurons of wt, Cln1-/- and Cln5-/- mice.B) Western blot analysis of cytoplasmic and membrane bound fractions of cytoskeletal, growth-cone and synapse assembly proteins. Antibodies: β-tubulin, synapsin 1, synapsin 1&2, Rab3 and Gap43.