Transcriptome profiles associated with neurodegeneration in the Engrailed-1 mouse model for Parkinson’s disease

Abstract

Parkinson’s disease (PD) is a highly heterogenous disorder characterized by bradykinesia, rigidity, rest tremor and non-motor symptoms like cognitive impairment, anosmia, and sleep disturbance. The underlying pathological hallmarks of PD are the degeneration of dopaminergic pigmented neurons in the substantia nigra pars compacta, neuroinflammation and, alpha synuclein inclusions accumulating into Lewy bodies and Lewy neurites in the substantia nigra. Polymorphisms in the Engrailed-1 (EN1) gene, a developmental gene of the homeobox family, have been associated with PD. Furthermore, studies have shown that the consequences of partial or complete knock-out of the mouse ortholog En1 differ between strains. The SwissOF1-En1+/− mouse strain shows loss of dopaminergic neurons in the substantia nigra whereas the C57Bl/6J-En1+/− strain lacks this neurodegenerative phenotype. To further investigate this phenomenon, RNA-sequence was carried out on 1-week old SwissOF1 and C57Bl/6J wildtype (WT) and En1+/− nigral tissues (n=3) to perform gene expression analysis. The analysis identified 165 differentially expressed genes between SwissOF1-En1+/- and WT; 89 between C57Bl/6J-En1+/- and WT; 211 between C57Bl/6J WT and SwissOF1 WT, and 104 between C57Bl/6J-En1+/- and SwissOF1-En1+/-. These later served as candidate genes for downstream analysis and an initial experimental verification. Lastly, an immunohistochemistry protocol was optimized for use with an anti-Pax6 antibody—preparing for the subsequent verification of the selected differentially expressed genes, following this project. In conclusion, the current project substantiated previous findings, found novel genes of interest and set them up for further investigation. Hopefully, these genes could elucidate PD etiology and further the understanding of dopaminergic cell death related to PD.

Popular Abstract

Genes may serve as neuroprotective agents in PD

Parkinson’s disease (PD) is one of the most common neurodegenerative disorders affecting more than 1% of people above 65 years. Although there is substantial knowledge about the course of the disease, the underlying causes has yet to be elucidated. Some genes are known to increase the risk of developing PD and the aim of this project was to find new genes that may play a role in PD susceptibility.

PD is an incurable disease which causes various motor and non-motor symptoms like resting tremor and body rigidity or sleep disturbance and fatigue. During PD, the neurons that produce/release dopamine start to degenerate in the brain region substantia nigra, and the protein alpha synuclein abnormally aggregates in these (dopaminergic) neurons. This protein aggregation initiates subsequent neurodegeneration which leads to the appearance of the disease associated symptoms. While we know that these neurons degenerate, the question of why remains unanswered. Many genes have been shown to increase the risk of developing PD, leading to the hypothesis that specific genes may affect the vulnerability of these dopaminergic neurons to the disease.

Animal models of PD, for example using mice, have been developed to mimic the disease process and make it easier to study its potential causes and progression. One example being the deletion (transgenic) of a copy of the Engrailed-1 (En1) gene in the SwissOF1 mouse. Interestingly, when the same partial gene deletion is introduced in another mouse strain (C57Bl/6JJ), these mice do not display degeneration of dopaminergic neurons. Although the mice from the two different strains share the same set of genes there are variations in the genetic code that e.g. affect the activity of the genes called gene expression. Since genes usually encode proteins, the suspected differences should be detectable on the gene expression level and on the protein level as well.

First, the gene expression in substantia nigra of mice from the two strains with and without partial the knock-out were compared. This analysis identified multiple genes that were differentially expressed between the different mouse strains. The biggest difference was found between the normal and En1-transgenic SwissOF1 mice. This means that the deletion of a copy of En1 resulted in bigger changes on the gene expression level for the SwissOF1 strain than for the C57Bl/6JJ strain. Three genes were then selected to be studied at the protein level. Brain tissues from studied mice were stained (selectively coloured) to show the location of the selected proteins. This way, the differences found on the gene expression level could be verified on the protein level.

Information about the genes found to be differentially expressed between the normal and En1-transgenic SwissOF1 mice was collected, revealing molecular processes that could be involved in the susceptibility to PD. Identifying the genes causing the transgenic C57Bl/6J mice to be resistant to the changes induced by the deletion of one copy of the En1 gene, in contrast to the other strain, may reveal that the increased or decreased expression of those genes can protect the dopaminergic neurons from degeneration. These findings can hopefully facilitate the development of new therapies for PD.

Master’s Degree Project in Molecular Biology 45 credits 2020
Department of Biology, Lund University

Advisor: Dr. Maria Swanberg
Translational Neurogenetics Unit, Department of Experimental Medical Science, Lund Universit

• author: Ralbovszki, Dorottya Maria
• supervisor: Maria Swanberg
• organization: Degree Projects in Molecular Biology
• course: MOBN02 20192
• year: 2020
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9031889
• date added to LUP: 2020-11-12 13:33:34
• date last changed: 2020-11-12 13:33:34