Abstract
INTRODUCTION
Parkinson’s disease (PD) is the second most frequent neurodegenerative disorder in aging. Mutations in the PRKN gene, encoding parkin, represent the most common known cause of autosomal recessive early-onset parkinsonism. The parkin protein is an E3 ubiquitin ligase that has been implicated in several cellular functions including the mitochondrial biogenesis activation, the mtDNA transcription modulation, the mitochondrial genome integrity, the mitophagy pathway, and the control of the cell´s apoptotic response during mitophagy. Furthermore, parkin was found to actively inhibit mitochondria-derived vesicle formation and mitochondrial antigen presentation (MitAP), and in parkin deficiency, inflammatory conditions trigger MitAP in immune cells. To further investigate its role in the pathogenesis of PD, human induced pluripotent stem cells (hiPSCs) were used to generate patient-specific dopaminergic neurons. However, the complexity of the human brain is not fully recapitulated by existing monolayer culture methods. Neurons differentiated in a three dimensional (3D) in vitro culture system might better mimic the in vivo cellular environment for basic mechanistic studies and represent better predictors of drug responses in vivo. In this study, targeted RNA-sequencing analysis in hiPSC-derived dopaminergic neurons cultured in 2D and 3D model systems were performed to investigate disease-linked mechanisms caused by PRKN mutations.
EXPERIMENTAL
Human iPSCs from two control individuals and two PD patients carrying a biallelic mutation in the PRKN gene (homozygous c.1072Tdel and homozygous Ex3 deletion, respectively) were included in the study. Midbrain dopaminergic (mDA) neurons were generated by using both a conventional 2D protocol (Kriks et al. 2011; Zanon et al., 2017) and a recently established hydrogel-based 3D system (Gilmozzi, Gentile et al., 2021). Samples for RNA isolation were collected at days 0, 10, 20 and 35 of differentiation. Targeted RNA-sequencing libraries were produced using the DriverMapT Human Genome-Wide Expression Profiling Kit, V2 (Cellecta). Differential expression analysis was performed with DESeq2 package (v 1.28.1) using the Wald test for significance testing. Functional annotation of differentially expressed genes (DEGs) was performed on KEGG, Reactome and Gene Ontology databases with ClusterProfiler (v3.16.1) and ReactomePA (v1.32.0).
RESULTS AND DISCUSSION
For both 2D and 3D protocols, a clear transition in gene expression from hiPSC markers (MYC and POU5F1-OCT3/4) to genes associated with mDA differentiation (PTCH1, FZDZ, HES1, OTX2, SLIT1, and LMX1A), and finally to an early expression of mature mDA markers (DCX and DDC) for all cell lines was observed. Overall, 1,426 genes were differentially expressed in PRKN mutation carriers compared to healthy individuals at day 35 (351 upregulated and 1,075 downregulated) in the 2D culture condition. Functional annotation analysis of DEGs altered exclusively at each timepoint highlighted cellular response to starvation, protein oxidation, synaptic transmission and axonogenesis as significantly enriched pathways. Additionally, 33 genes were upregulated and 118 genes were downregulated in the PRKN mutant cell lines compared to controls at all timepoints. Functional annotation analysis of these genes indicated that the immune response pathways are significantly enriched in the PRKN mutants compared to the controls. By analysing the 3D generated transcriptome, 57 genes were differentially expressed in PRKN mutation carriers compared to healthy individuals at day 35 (21 upregulated and 36 downregulated). Functional annotation analysis of DEGs altered exclusively at each timepoint showed that immune response pathways, regulation of lipid metabolism, and regulation of insulin-like growth factor were significantly enriched. In this dataset, 12 genes were upregulated and 23 genes were downregulated in the PRKN mutant cell lines compared to the controls at all timepoints. Enrichment analysis of these DEGs confirmed a strong correlation between the PRKN gene mutation and the alteration of the immune response pathways and inhibition of insulin secretion.
CONCLUSION
Overall, an involvement of dysfunctional parkin in altered axonogenesis, mitochondrial metabolism, and immune response pathways was assessed by using two differentiation approaches. By using the hydrogel 3D model system, we identified PRKN mutation-specific gene sets and functional pathways linked to the regulation of insulin-like growth factor and lipid metabolism, thus highlighting the involvement of parkin in these pathways and providing new hypotheses for the development and progression of PD.