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Whole-genome gene expression and splicing analysis of midbrain neurons derived from human embryonic stem cells: relevance to Parkinson's disease

ABSTRACT: Directed differentiation of midbrain dopaminergic neurons from human embryonic stem cells (hESCs) has galvanized much interest into their potential application in human Parkinson’s disease (PD). We conducted genome-wide, exon-specific expression analyses at three temporally and phenotypically distinct stages of lineage restriction (pluripotent hESCs, multipotent neural precursor cells and terminally differentiated midbrain dopaminergic neurons). We compare these to expression data generated on the same platform from samples isolated from human fetal brain and from human control postmortem samples isolated from the substantia nigra. This comparison highlights the commonalities and differences between neural cells derived from hESCs and their counterparts in the human brain. This gene expression microarray study was carried out to i) identify changes in gene expression and splicing during neural differentiation to dopaminergic neurons, and ii) determine the maturational state of hESC-derived neuronal samples particularly with regard to genes and pathways relevant to Parkinson's disease.

ORGANISM(S): Homo sapiens

PROVIDER: GSE34865 | GEO | 2014/12/01

SECONDARY ACCESSION(S): PRJNA218467

REPOSITORIES: GEO

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Project description:Mutations in the SNCA gene cause autosomal dominant Parkinson’s disease (PD), with progressive loss of dopaminergic neurons in the substantia nigra, and accumulation of aggregates of α-synuclein. However, the sequence of molecular events that proceed from the SNCA mutation during development, to its end stage pathology is unknown. Utilising human induced pluripotent stem cells (hiPSCs) with SNCA mutations, we resolved the temporal sequence of pathophysiological events that occur during neuronal differentiation in order to discover the early, and likely causative, events in synucleinopathies. We adapted a small molecule-based protocol that generates highly enriched midbrain dopaminergic (mDA) neurons (>80%). We characterised their molecular identity using single-cell RNA sequencing and their functional identity through the synthesis and secretion of dopamine, the ability to generate action potentials, and form functional synapses and networks. RNA velocity analyses confirmed the developmental transcriptomic trajectory of midbrain neural precursors into different mDA neuronal clusters. To characterise the synucleinopathy, we adopted super-resolution methods to determine the number, size, and structure of aggregates in SNCA-mutant mDA neurons. By day 27 of differentiation, prior to maturation to mDA neurons of molecular and functional identity, we demonstrate the formation of small aggregates; specifically, β-sheet rich oligomeric aggregates, in SNCA-mutant midbrain immature neurons. The aggregation progresses over time to accumulate phosphorylated and fibrillar aggregates. When the midbrain neurons were functional, we observed impaired intracellular calcium signalling, evidenced with an increased basal calcium level and impairments in both cytosolic and mitochondrial calcium rearrangements. Once midbrain identity fully developed, SNCA-mutant neurons exhibited mitochondrial dysfunction, oxidative stress, lysosomal swelling as well as an upregulation of mitophagy and autophagy. In addition, SNCA-mutant neurons displayed pathophysiological excitability, revealed as a depolarised resting membrane potential, an increased input resistance, and impaired firing properties. Ultimately these multiple cellular stresses lead to an increase in cell death by day 62 post-differentiation. Our differentiation paradigm generates an efficient model for studying disease mechanisms in PD, and highlights that protein misfolding to generate intraneuronal oligomers is one of the earliest critical events driving disease in human neurons, rather than a late-stage hallmark of the disease.

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Whole-genome gene expression and splicing analysis of midbrain neurons derived from human embryonic stem cells: relevance to Parkinson's disease

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