Nigral transcriptomic profiles in Engrailed-1 hemizygous mouse models of Parkinson's disease reveal upregulation of oxidative phosphorylation-related genes associated with delayed dopaminergic neurodegeneration

Abstract

Introduction: Parkinson's disease (PD) is the second most common neurodegenerative disorder, increasing both in terms of prevalence and incidence. To date, only symptomatic treatment is available, highlighting the need to increase knowledge on disease etiology in order to develop new therapeutic strategies. Hemizygosity for the gene Engrailed-1 (En1), encoding a conserved transcription factor essential for the programming, survival, and maintenance of midbrain dopaminergic neurons, leads to progressive nigrostriatal degeneration, motor impairment and depressive-like behavior in SwissOF1 (OF1-En1^+/-). The neurodegenerative phenotype is, however, absent in C57Bl/6j (C57-En1^+/-) mice. En1^+/- mice are thus highly relevant tools to identify genetic factors underlying PD susceptibility.

Methods: Transcriptome profiles were defined by RNAseq in microdissected substantia nigra from 1-week old OF1, OF1- En1^+/-, C57 and C57- En1^+/- male mice. Differentially expressed genes (DEGs) were analyzed for functional enrichment. Neurodegeneration was assessed in 4- and 16-week old mice by histology.

Results: Nigrostriatal neurodegeneration was manifested in OF1- En1^+/- mice by increased dopaminergic striatal axonal swellings from 4 to 16 weeks and decreased number of dopaminergic neurons in the SNpc at 16 weeks compared to OF1. In contrast, C57- En1^+/- mice had no significant increase in axonal swellings or cell loss in SNpc at 16 weeks. Transcriptomic analyses identified 198 DEGs between OF1- En1^+/- and OF1 mice but only 52 DEGs between C57- En1^+/- and C57 mice. Enrichment analysis of DEGs revealed that the neuroprotective phenotype of C57- En1^+/- mice was associated with a higher expression of oxidative phosphorylation-related genes compared to both C57 and OF1- En1^+/- mice.

Discussion: Our results suggest that increased expression of genes encoding mitochondrial proteins before the onset of neurodegeneration is associated with increased resistance to PD-like nigrostriatal neurodegeneration. This highlights the importance of genetic background in PD models, how different strains can be used to model clinical and sub-clinical pathologies and provides insights to gene expression mechanisms associated with PD susceptibility and progression.

Keywords: Parkinson’s disease; genetic susceptibility; mitochondria; neurodegeneration; substantia nigra; transcriptomics.

Figure 1

Analysis of genetic background effects on neurodegeneration in the Engrailed 1 hemizygous (En1^+/−) mouse model for Parkinson’s disease (PD). (A) Study design and experimental setup (n = 3). (B) Sections used for histological analyses of PD-like neuropathology in En1^+/− mice. (C) Methodology for bulk RNA-Seq analysis of laser capture microdissected substantia nigra pars compacta (SNpc) from 1 week-old mice. OF1: SwissOF1; C57, C57Bl/6J; TH, tyrosine hydroxylase.

Figure 2

Engrailed 1 hemizygosity (En1^+/−) induces progressive nigrostriatal dopaminergic neurodegeneration in OF1 but not C57 mice. (A) Representative images of axonal swellings in the striatum of OF1-En1^+/− and C57-En1^+/− mice at 4 and 16 weeks-of-age. (B) In OF1-En1^+/−mice, there is a significant increase in the number of swellings per remaining dopaminergic neuron in the substantia nigra pars compacta (SNpc) between 4 and 16 weeks, while axonal swellings do not increase in C57-En1^+/− mice. Data presented as mean ± SD (Two-way ANOVA with Tukey’s multiple comparison test, n = 7). (C) Representative images of SNpc from OF1 wild type (WT) and OF1-En1^+/− mice at 4 and 16 weeks-of-age. (D) The number of dopaminergic neurons in the SNpc is significantly lower in 16 week-old OF1-En1^+/− mice compared to both 16 week-old OF1 WT and 4 week-old OF1-En1^+/− mice. (E) Representative images of SNpc from C57 WT and C57-En1^+/− mice at 4 and 16 weeks-of-age. (F) C57-En1^+/− mice did not display significant loss of dopaminergic neurons in the SNpc at 16 weeks. TH, tyrosine hydroxylase; OF1, SwissOF1; C57, C57Bl/6J.

Figure 3

Engrailed-1 hemizygosity (En1^+/−) effects on nigral transcriptomic profiles in OF1 and C57 mice. (A) Volcano plots showing all expressed genes in comparisons OF1-En1^+/−vs. OF1 wild-type (WT) mice and C57- En1^+/− vs. C57 WT mice. Vertical lines indicate fold change (|FC|) of 1.5. (B) Venn diagram illustrating differentially expressed genes (DEGs) in OF1-En1^+/− vs. OF1 WT and in C57-En1^+/− vs. C57 WT, overlapping only by 2 DEGs. (C) Gene Ontology (GO) enrichment analysis of DEGs in OF1-En1^+/− vs. OF1 WT mice. (D) Reactome pathway analysis for DEGs in OF1-En1^+/− vs. OF1 WT mice. (E) Heatmap of enriched pathway genes in OF1-En1^+/− vs. OF1 WT mice. OF1-En1^+/− mice show a higher expression of genes associated with neuron development and axonal guidance. (F) GO enrichment analysis of DEGs in C57-En1^+/− vs. C57 WT mice. (G) Reactome pathway analysis for DEGs in C57-En1^+/− vs. C57 WT mice. (H) KEGG pathway enrichment analysis for DEGs in C57-En1^+/− vs. C57 WT mice, including Parkinson’s disease. (I) Heatmap of enriched pathway genes in C57-En1^+/− vs. C57 WT mice. C57- En1^+/− mice show a higher expression of genes encoding mitochondrial proteins (marked with *). For gene symbols, mt stands for genes encoded by the mitochondrial genome. Red: up-regulated, blue: down-regulated. GO terms were separated according to their Ontology category (Biological Process, Cellular Compartment and Molecular Function). OF1, SwissOF1; C57, C57Bl/6J.

Figure 4

Transcriptomic differences between OF1 wild-type (WT) and C57 WT mice as well as OF1 Engrailed-1 hemizygous (En1^+/−) and C57-En1^+/− mice. (A) Volcano plots showing all expressed genes in OF1 WT vs. C57 WT mice and in OF1-En1^+/− vs. C57-En1^+/− mice. Vertical lines indicate fold change (FC) of 1.5. (B) Venn diagram differentially expressed genes (DEGs) in OF1 WT vs. C57 WT and in OF1-En1^+/− vs. C57-En1^+/− mice. (C) Gene Ontology (GO) enrichment analysis of DEGs in OF1 WT vs. C57 WT mice. (D) Reactome pathway analysis for DEGs in OF1 WT vs. C57 WT mice shows pathways associated with vesicular trafficking and antigen presentation. (E) Heatmap of enriched pathway genes in OF1 WT vs. C57 WT mice. OF1 WT mice show a higher expression of genes associated with antigen presentation and vesicle trafficking. (F) GO enrichment analysis of the 142 DEGs exclusive to OF1-En1^+/− vs. C57-En1^+/− mice shows enrichment in GO terms associated to energy metabolism in mitochondria. (G) Heatmap of enriched pathway genes in OF1-En1^+/− vs. C57-En1^+/− mice. C57-En1^+/− mice consistently show higher expression of genes associated with mitochondrial respiration and oxidative phosphorylation (marked with*). For gene symbols, mt stands for genes encoded by the mitochondrial genome. Red: up-regulated, blue: down-regulated. GO terms were separated according to their Ontology category (Biological Process, Cellular Compartment and Molecular Function). OF1, SwissOF1; C57, C57Bl/6J.

Figure 5

C57 hemizygous (En1^+/−) mice have higher expression of genes encoding components of the oxidative phosphorylation (OxPhos) pathway compared to OF1 En1^+/− and C57 wild-type (WT) mice. (A) KEGGs representative diagram of protein complexes in the OxPhos pathway located in the mitochondrial inner membrane. (B) Normalized counts of regulated transcripts mapped to the OxPhos-pathway. C57-En1^+/− mice have higher expression of genes coding for complex I and V subunits compared to OF1-En1^+/− mice. C57- En1^+/− mice also have higher expression of genes coding for components of complex III compared to C57 WT mice and C57 WT mice have higher expression of mtAtp8 (in complex V) compared to OF1 WT mice. OF1, SwissOF1; C57, C57Bl/6J. (Figure 5A) modified from KEGG: Kyoto Encyclopedia of Genes and Genomes, map00190.