Genetically Encoded and Modular SubCellular Organelle Probes (GEM-SCOPe) reveal lysosomal and mitochondrial dysfunction driven by PRKN knockout.

Cellular processes including lysosomal and mitochondrial dysfunction are implicated in the development of many diseases. Quantitative visualization of mitochondria and lysosoesl is crucial to understand how these organelles are dysregulated during disease. To address a gap in live-imaging tools, we developed GEM-SCOPe (Genetically Encoded and Modular SubCellular Organelle Probes), a modular toolbox of fluorescent markers designed to inform on localization, distribution, turnover, and oxidative stress of specific organelles. We expressed GEM-SCOPe in differentiated astrocytes and neurons from a human pluripotent stem cell PRKN-knockout model of Parkinson's disease and identified disease-associated changes in proliferation, lysosomal distribution, mitochondrial transport and turnover, and reactive oxygen species. We demonstrate GEM-SCOPe is a powerful panel that provide critical insight into the subcellular mechanisms underlying Parkinson's disease in human cells. GEM-SCOPe can be expanded upon and applied to a diversity of cellular models to glean an understanding of the mechanisms that promote disease onset and progression.

Evaluation of two transposases for improving expression of recombinant proteins in Chinese hamster ovary cell stable pools by co-transfection and supertransfection approaches.

Transposons are genetic elements capable of cutting and pasting genes of interest via the action of a transposase and offer many advantages over random or targeted integration of DNA in the creation of Chinese hamster ovary (CHO) cell lines for recombinant protein expression. Unique transposases have different recognition sites, allowing multiple transposases to be co-transfected together. They also allow for supertransfection (transfection on a previously transfected pool or cell line) with a second transposase to integrate additional copies of the same gene or an additional gene without disruption of the previously integrated DNA which to our knowledge has not been previously described in literature. Two fluorescent proteins, EGFP and tagRFP657, were either co-transfected or supertransfected into CHO cells using two unique transposases and showed high expression efficiency with similar expression levels (measured as mean fluorescence intensity), regardless of whether the genes were co-transfected or supertransfected onto an existing stable pool. Additionally, dual selection of the genes, both in the absence of L-glutamine and the presence of puromycin, led to higher expression levels than single selection alone. These results demonstrate that supertransfection using unique transposases could be a useful strategy for increasing titers of existing cell lines or for overexpressing helper (non-therapeutic) genes to improve expression and/or product quality of existing pools and cell lines, potentially saving significant time and resources.

Molecular profiling of human substantia nigra identifies diverse neuron types associated with vulnerability in Parkinson's disease.

Parkinson's disease (PD) is characterized pathologically by the loss of dopaminergic (DA) neurons in the substantia nigra (SN). Whether cell types beyond DA neurons in the SN show vulnerability in PD remains unclear. Through transcriptomic profiling of 315,867 high-quality single nuclei in the SN from individuals with and without PD, we identified cell clusters representing various neuron types, glia, endothelial cells, pericytes, fibroblasts, and T cells and investigated cell type-dependent alterations in gene expression in PD. Notably, a unique neuron cluster marked by the expression of RIT2, a PD risk gene, also displayed vulnerability in PD. We validated RIT2-enriched neurons in midbrain organoids and the mouse SN. Our results demonstrated distinct transcriptomic signatures of the RIT2-enriched neurons in the human SN and implicated reduced RIT2 expression in the pathogenesis of PD. Our study sheds light on the diversity of cell types, including DA neurons, in the SN and the complexity of molecular and cellular changes associated with PD pathogenesis.

Disruption of lysosomal proteolysis in astrocytes facilitates midbrain organoid proteostasis failure in an early-onset Parkinson's disease model.

Accumulation of advanced glycation end products (AGEs) on biopolymers accompanies cellular aging and drives poorly understood disease processes. Here, we studied how AGEs contribute to development of early onset Parkinson's Disease (PD) caused by loss-of-function of DJ1, a protein deglycase. In induced pluripotent stem cell (iPSC)-derived midbrain organoid models deficient for DJ1 activity, we find that lysosomal proteolysis is impaired, causing AGEs to accumulate, α-synuclein (α-syn) phosphorylation to increase, and proteins to aggregate. We demonstrated these processes are at least partly driven by astrocytes, as DJ1 loss reduces their capacity to provide metabolic support and triggers acquisition of a pro-inflammatory phenotype. Consistently, in co-cultures, we find that DJ1-expressing astrocytes are able to reverse the proteolysis deficits of DJ1 knockout midbrain neurons. In conclusion, astrocytes' capacity to clear toxic damaged proteins is critical to preserve neuronal function and their dysfunction contributes to the neurodegeneration observed in a DJ1 loss-of-function PD model.

Novel human pluripotent stem cell-derived hypothalamus organoids demonstrate cellular diversity.

The hypothalamus is a region of the brain that plays an important role in regulating body functions and behaviors. There is a growing interest in human pluripotent stem cells (hPSCs) for modeling diseases that affect the hypothalamus. Here, we established an hPSC-derived hypothalamus organoid differentiation protocol to model the cellular diversity of this brain region. Using an hPSC line with a tyrosine hydroxylase (TH)-TdTomato reporter for dopaminergic neurons (DNs) and other TH-expressing cells, we interrogated DN-specific pathways and functions in electrophysiologically active hypothalamus organoids. Single-cell RNA sequencing (scRNA-seq) revealed diverse neuronal and non-neuronal cell types in mature hypothalamus organoids. We identified several molecularly distinct hypothalamic DN subtypes that demonstrated different developmental maturities. Our in vitro 3D hypothalamus differentiation protocol can be used to study the development of this critical brain structure and can be applied to disease modeling to generate novel therapeutic approaches for disorders centered around the hypothalamus.

High-throughput generation of midbrain dopaminergic neuron organoids from reporter human pluripotent stem cells.

Here, we describe a high-throughput 3D differentiation protocol for deriving midbrain dopaminergic neurons from human pluripotent stem cells. The use of organoids has become prevalent in disease modeling, but there is a high demand for more homogeneous cultures. Our approach is advantageous for large-scale production of uniform midbrain organoids that can be maintained in diverse formats, and our reporters allow for sorting of dopaminergic neurons. The maturing long-term organoid cultures can be used as a model for the entire midbrain. For complete details on the use and execution of this protocol, please refer to Ahfeldt et al. (2020).

Pathogenic Pathways in Early-Onset Autosomal Recessive Parkinson's Disease Discovered Using Isogenic Human Dopaminergic Neurons.

Parkinson's disease (PD) is a complex and highly variable neurodegenerative disease. Familial PD is caused by mutations in several genes with diverse and mostly unknown functions. It is unclear how dysregulation of these genes results in the relatively selective death of nigral dopaminergic neurons (DNs). To address this question, we modeled PD by knocking out the PD genes PARKIN (PRKN), DJ-1 (PARK7), and ATP13A2 (PARK9) in independent isogenic human pluripotent stem cell (hPSC) lines. We found increased levels of oxidative stress in all PD lines. Increased death of DNs upon differentiation was found only in the PARKIN knockout line. Using quantitative proteomics, we observed dysregulation of mitochondrial and lysosomal function in all of the lines, as well as common and distinct molecular defects caused by the different PD genes. Our results suggest that precise delineation of PD subtypes will require evaluation of molecular and clinical data.