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Life With Parkinson’s Disease – Single Cell Sequencing Paves the Path to Cell Replacement Therapies


[↓] Animal in vitro Models of PD

[↓] Human in vitro Models of PD

[↓] Human Postmortem Substantia Nigra

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, effecting 1% of the population above the age of 60. Global incidence and prevalence of PD increases each year, potentiating scientific and clinical efforts to combat PD and to understand molecular mechanisms of the disease. PD is characterized by a loss of dopaminergic neurons (DaNs) in the substantia nigra pars compacta (SNpc), which results in symptoms such as rigidity, postural instability, tremor at rest, and bradykinesia. Histologically, PD features accumulation of small and complex structures called Lewy bodies (LBs) which contain aggregated forms of α-synuclein (α-syn), including fibrils.

Single cell RNA sequencing technologies enabled the assessment of cell heterogeneity and reconstruction of the temporal and spatial dynamics of complex tissues. Although neuroscientists efficiently leveraged several multiomics approaches to study other diseases, they only recently started applying them to study PD in human postmortem tissues due to technology and RNA preservation limitations. Applying multiple modality sequencing analysis of animal and human in vitro PD models on the single cell level has transformed our understanding of the heterogenous cellular composition and diversity of neuronal and glial cell type identities in the developing mouse and human brain. This led to identifying their functional role in the DaN degenerative process underlying PD.

Genetic-based models of PD and parkinsonism have involved familial PD-associated mutant forms of SNCA (α-syn), overexpression or knock-in mutations of LRRK2 or deletion or knock-out of PRKN or PINK1. Preformed α-syn fibrils can be injected into the striatum or substantia nigra (SN) using animal models for the development of Lewy-like α-syn fibrillar inclusions and aggregates that closely recapitulate features of human PD. Single cell profiles can be obtained via single cell sequencing and multiomics to construct interactomes or trajectory maps for a more holistic view on the disease. Here are several examples of single cell sequencing implementation in animal and human models of PD and parkinsonism that open the door for development of cell replacement therapies.

Animal in vitro Models of PD [↑]

The earliest attempts to characterize DaN development and diversity through single cell sequencing analysis used mouse embryos and early postnatal DaNs. La Manno et al. performed integrative analysis of scRNA-seq data derived from ventral midbrain in human and mouse and identified specific adult dopaminergic cell types that emerged postnatally, and several diverse radial glia-like cell types that biased toward a distinct fate. [1] This study faithfully depicted the degree to which species differ in developmental timing and cell proliferation in the context of molecular diversity. Tiklova et al. performed network analyses and identified seven neuronal subpopulations divided into two major branches, revealing novel cellular populations that are developmentally related but are non-dopaminergic. [2] These studies have important implications for developing a cell therapy strategy for PD.

Figure 1. scRNA-seq of mouse neural progenitors from embryos and early postnatal DaNs. (Left) [1] t-SNE plot of cells colored by cell type. (Right) [2] Network plot depicting distribution of Pitx3-expressing midbrain neurons colored by molecularly defined cell type.

Human in vitro Models of PD [↑]

Induced pluripotent stem cell (iPSC) technology revolutionized our ability to model neurodegenerative diseases, generating patient-derived in vitro neurons otherwise inaccessible to neuroscientists, allowing insights into the early disease formation as opposed to the end stages represented in postmortem brain analyses. Differentiating neuronal stem cells into DaNs represents the earliest stages of the disease process, facilitating discovery of novel biomarkers and therapeutic candidates. Regardless of the midbrain patterning protocol, DaN cultures can be highly heterogeneous, with different cell subtypes adding layers of complexity and inconsistencies in downstream analyses.

To overcome these limitations, Lang et al. performed single cell RNA sequencing using hiPSC-derived DaNs from controls and PD GBA-N370S patients and revealed a functionally enriched gene set that defined a pseudo-temporal axis of gene expression variation in mutant hiPSC-derived DaN. [3]Fernandes et al. explored cell type-specific responses to PD-relevant stress-induced perturbations of these cells and identified six distinct cell types, including two neuron progenetic populations expressing dopaminergic progenitor markers. [4] Finally, derivation of stem cells from embryonic origins allows for cell engraftment of hiPSC-derived functional neurons or progenitors into PD patients, [5] paving the path for cell replacement therapy development.

Figure 2. scRNA-seq of human induced pluripotent stem cell (hiPSC)-derived neurons of PD patients. [4] UMAP of dissociated cells after WT iPSC-dopaminergic neuron differentiation.

Human Postmortem Substantia Nigra [↑]

Recent effort to profile individual nuclei from human postmortem brain tissues have demonstrated efficient classification of cell types and spatiotemporal dynamics of cellular compositions at single cell resolution. For example, Darmanis et al. conducted a survey of human neocortex transcriptome diversity and identified novel subpopulations of adult neurons that expressed major histocompatibility complex type I genes, in which such an expression pattern was not observed in fetal neurons. [6] By combining bulk tissue RNA-seq and scRNA-seq, Liu et al. further profiled RNA from human neocortex at different stages of development and identified specific target enrichment in radial glia cells but not in tissues. [7]

Such studies have constructed open-source single cell RNA sequencing databases such as those for mid-gestation human neocortex, substantia nigra and the Allen Brain Cell Types Database containing primary motor cortex, middle temporal gyrus, primary visual cortex, and anterior cingulate cortex. For example, human single-nucleus transcriptomic atlas for the substantia nigra (SN) have identified cell clusters spanning known resident cell classes, including astrocytes (ASC), oligodendrocytes (ODC), oligodendrocyte progenitor cells (OPC), mural cells (endothelial cells and pericytes), microglia, fibroblasts, and neurons, including DaNs and multiple inhibitory types. [8,9]

Figure 3. The snRNA-seq of human postmortem brain tissues in studies of PD. UMAP plots colored by cell type. (Left) [8] (Right) [9]

References [↑]

[1] La Manno et al. Molecular Diversity of Midbrain Development in Mouse, Human, and Stem Cells. Cell, 2016. [PubMed]

[2] Tiklova et al. Single-cell RNA sequencing reveals midbrain dopamine neuron diversity emerging during mouse brain development. Nat. Commun., 2019. [PubMed]

[3] Lang et al. Single-Cell Sequencing of iPSC-Dopamine Neurons Reconstructs Disease Progression and Identifies HDAC4 as a Regulator of Parkinson Cell Phenotypes. Cell Stem Cell 2019. [PubMed]

[4] Fernandes et al. Single-Cell Transcriptomics of Parkinson’s Disease Human In Vitro Models Reveals Dopamine Neuron-Specific Stress Responses. Cell Rep. 2020. [PubMed]

[5] Schweitzer et al. Personalized iPSC-Derived Dopamine Progenitor Cells for Parkinson’s Disease. N. Engl. J. Med. 2020. [PubMed]

[6] Darmanis et al. Survey of human brain transcriptome diversity at the single cell level. Proc. Natl. Acad. Sci. 2015. [PubMed]

[7] Liu et al. Single-cell analysis of long non-coding RNAs in the developing human neocortex. Genome Biol. 2016. [PubMed]

[8] Agarwal et al. A single-cell atlas of the human substantia nigra reveals cell-specific pathways associated with neurological disorders. Nat. Commun. 2020. [PubMed]

[9] Welch et al. Single-Cell Multi-omic Integration Compares and Contrasts Features of Brain Cell Identity. Cell 2019. [PubMed]