(H-310) Evaluating the Single Cell Transcriptomic Profiles of an iPSC-derived Neuron-Astrocyte Assembloid Exposed to Tauopathy Vs. Synucleinopathy

Introduction:: The modes of degeneration varies immensely across the hundreds of neurodegenerative diseases seen clinically today, however protein misfolding remains one of the most prominent precursory pathologies. Tauopathies are characterized by the accumulation of misfolded or hyperphosphorylated tau aggregates while synucleinopathies are associated with the formation of insoluble α-synuclein aggregates - in both cases protein aggregation precipitates neurodegeneration. Human induced pluripotent stem cell (hiPSC) models are gaining widespread usage due to their ability to better recapitulate human physiology however, 2D neuronal models show limited arborization and cell-cell interactions as well as lack the extensive pathology and degeneration typically associated with these diseases. Further, current 3D models require months or years to mature and also display limited pathology and neurodegeneration. The Wolozin lab has developed a method to produce an hiPSC-derived 3D “assembloid” composed of neurons (N) and astrocytes (A) that, when seeded with tau oligomers (oTau) or α-synuclein pre-formed fibrils (αsPFFs), rapidly recapitulates the respective pathology as well as neuron-neuron and neuron-glia interactions. This model is able to generate robust tau or synuclein pathology, neurodegeneration, and reactive astrogliosis over just three weeks of culture. In this study, tauopathy and synucleinopathy were induced in iPS-N/A assembloids and analyzed using single cell RNA sequencing (scRNA-seq) to elucidate differences in the propagation of neurodegeneration across these two disease types. Understanding the specific transcriptomic changes that take place as a result of a particular pathology could prove a strong tool for drug discovery across many neurodegenerative diseases.

Materials and Methods::

Patient-derived iPSCs (APP V717I) and control hiPSCs were thawed, maintained in mTeSRTM Plus culture, and then differentiated to neuronal progenitor cells (NPCs) following STEMCELLTM protocols.NPCs were maintained in serum-free STEMdiffTM Neural Progenitor Medium 2 and all NPCs used for these experiments were kept at passage < 6. NPCs were plated in STEMdiffTM Forebrain Neuron Differentiation Media on Corning® Matrigel®-coated tissue culture treated plates and transduced with a NEUROG2 lentivirus to induce iPS-N. Similarly, iPS-A were produced when NPCs were passaged at 90% confluence and reseeded in STEMdiffTM Astrocyte Differentiation Media. oTau was generated from frozen hippocampus and cortical tissue of 9 month old PS19 P301S tau transgenic mice over-expressing human Tau. αsPFFs were generated using standard protocol. iPS-N were selectively exposed to 0.04 mg/mL oTau or 1µg/mL αsPFFs directly in cell culture for 24h before incorporation into assembloid culture.

iPS-N and iPS-A were combined in a single cell suspension in a 1:1 ratio in assembloid media (DMEM/F12, 1% Glutamax, 1% Sodium Pyruvate, 1% N-2 Supplement, 1% B-27 Supplement, 10 uM Y-27632, 1% PenStrep, and 1 mg/mL Heparin) and plated in AggreWellTM800 microwells coated with Anti-Adherence Rinsing Solution. At one week, the iPS-N/A assembloids were transferred to ultra-low attachment round-bottom 96-well plates and maintained in 100-200µL assembled media rotating at 85 rpm. 

The assembloids were then harvested and each conditions were combined in a single cell suspension. A cDNA library was generated and then sequenced on Illumina’s NextSeq 500 or NOVAseq. CellRanger was used to align reads and find barcodes.

Results, Conclusions, and Discussions:: The iPS-N/A assembloid model developed by the Wolozin lab is a reproducible model of protein-opathy reaching maturity in three weeks. This system specifically allows for the analysis of pathology progression and subsequent neurodegenerative processes while simultaneously analyzing astrocytes and neuroinflammation. Notably, the astrocyte response to tauopathy was reminiscent of that of the Alzehimer's Disease (AD) human brain with astrogliosis marker GFAP and astrocyte marker S100β both showing statistically significant increases with pathology after both 14 and 21 days. Immunolabeling verified that each of the differentiated cell types showed their characteristic markers (Tuj, VGLUT1, GAD67, VIM). We also see cell-specific responses: the excitatory neurons decrease while the inhibitory neurons are less impacted by degeneration. Additionally, each of these cell types along with astrocytes has a unique transcript profile across conditions. Interestingly, analysis of GO pathways illustrated stark differences between Tau and α-synuclein pathology as can be seen in Figure 1A and B. Genes which were upregulated in the tauopathy environment were downregulated in the synucleinopathy environment (Fig. 1A), indicating that the mechanism by which these two proteins cause neuronal death and what effect each has on other cell machinery is mechanistically different. While each entails protein aggregation, each one also affects an entirely different set of transcripts downstream. Figure 1C details transcriptome changes due to synucleinopathy, which looks drastically different than those changes due to tauopathy in Figure 1A. These differences could be vital to the future of drug discovery for both of these pathologies. Future work will include the addition of microglia and vasculature to closer recapitulate human physiology as well as seeking out updated astrocyte culture methods that may allow for better electrophysiological maturation. Furthermore, discovering what effect pathological proteins have on the transcriptome can be applied to many other neurodegenerative or genetic conditions that may be robustly and reproducibly studied in this iPSC-N/A model and has the potential to elucidate disease-treating inhibitors to counteract these pathology-specific transcript changes.

Acknowledgements (Optional): : Special thanks to Hannah Rickner and Lushuang Zhang, recent alumni of the Wolozin lab without whom this work would not be possible.

References (Optional): : Rickner HD, Jiang L, Hong R, O'Neill NK, Mojica CA, Snyder BJ, Zhang L, Shaw D, Medalla M, Wolozin B, Cheng CS. Single cell transcriptomic profiling of a neuron-astrocyte assembloid tauopathy model. Nat Commun. 2022 Oct 21;13(1):6275. doi: 10.1038/s41467-022-34005-1. PMID: 36271092; PMCID: PMC9587045.

Galpern WR, Lang AE. Interface between tauopathies and synucleinopathies: a tale of two proteins. Ann Neurol. 2006 Mar;59(3):449-58. doi: 10.1002/ana.20819. PMID: 16489609.

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