cell inclusion survival tracking  (Nikon)

Journal: Neuron

Article Title: Rapid iPSC inclusionopathy models shed light on formation, consequence, and molecular subtype of α-synuclein inclusions

doi: 10.1016/j.neuron.2024.06.002

Figure Lengend Snippet: (A) Top, time-lapse imaging of LipidSpot(+) inclusions pre- (T = 0 min) and post-treatment (T = 60 min) with DMSO or trifluoperazine. Bottom, quantification of LipidSpot(+), LipidSpot(−) neurite-type, or soma-type inclusions at T = 60 min post-treatment (3 independent replicates per condition, reproduced across 3 neuronal differentiations using the highest treatment concentrations). (B) Time-lapse imaging in seeded PFF model capturing interaction between LipidSpot(+) inclusions and elongating neurite-type inclusion in the same cell. White arrow, neurite-type inclusion (GFP(+)/LipidSpot(−)). Inset, cell soma with two GFP(+)/LipidSpot(+) inclusions (DIV20), which become one GFP(+)/LipidSpot(−) inclusion (DIV29). (C) Confocal image of adjacent LipidSpot(+) and LipidSpot(−) inclusions. (D) Dynamic lattice light-sheet microscopy (3D rendering) of a LipidSpot(+)/GFP(+) soma-type inclusion. White arrows, small αS-sfGFP aggregates; pink arrowheads, sequestered lipid accumulations; pink arrow, lipid aggregate partially internalized into the inclusion. (E) pS129 and neurofilament IF in sporadic PD brain (frontal cortex) shows soma-type inclusion reminiscent of fusion examples in (B)–(D). (F) CLEM example of αS(+) inclusions with mixed amyloid and lipid pathology in substantia nigra of sporadic PD brain (top) and seeded inclusionopathy model (bottom). (G) Manual longitudinal single-inclusion and single-cell survival tracking. Log rank test: **** p < 0.0001. Error bars = SD.

Article Snippet: We developed algorithms for single-cell inclusion survival tracking with longitudinal imaging (BioStation CT, Nikon) for both spontaneous and seeded inclusionopathy models ( – ).

Techniques: Imaging, Microscopy

Journal: Neuron

Article Title: Rapid iPSC inclusionopathy models shed light on formation, consequence, and molecular subtype of α-synuclein inclusions

doi: 10.1016/j.neuron.2024.06.002

Figure Lengend Snippet: (A) Schematic of spontaneous inclusionopathy model. (B) Left, pS129 IF in pi-Ns overexpressing sfGFP-tagged or untagged αS-3K, untagged αS-WT, or sfGFP control. Right, quantification of IF. One-way ANOVA plus Tukey’s multiple comparison test: **** p < 0.0001; ns, not significant. (C) Western blot after sequential TX-100/SDS extraction of soluble and insoluble protein fractions. (D) CLEM for αS-pS129 (leftmost 3 panels) and LipidSpot labeling (right 2 panels) in spontaneous inclusionopathy model. (E) Immunoreactivity profiles of soma-type inclusions in pi-N 3K-sfGFP-pB model. Total number of inclusions counted shown in parentheses. (F) Inclusion subtype classification in spontaneous inclusionopathy model. (G) Inclusion survival tracking in spontaneous inclusionopathy model. Examples of GFP(+)/RFP(+) neurons detected in the BioStation CT. (H) Experimental time line for BioStation CT imaging. (I) Example of automated mask for soma-type inclusions. Neuron was identified as dead at the 90 h time point. (J) Kaplan-Meier curve comparing survival probabilities of pi-N 3K-sfGFP-pB model with and without inclusions and pi-N sfGFP-pB control neurons. Log rank test: * p < 0.05, *** p < 0.001, **** p < 0.0001. Data are representative of 3 separate neuronal differentiations. Error bars in (B) = SD.

Article Snippet: We developed algorithms for single-cell inclusion survival tracking with longitudinal imaging (BioStation CT, Nikon) for both spontaneous and seeded inclusionopathy models ( – ).

Techniques: Control, Comparison, Western Blot, Extraction, Labeling, Imaging

Journal: Neuron

Article Title: Rapid iPSC inclusionopathy models shed light on formation, consequence, and molecular subtype of α-synuclein inclusions

doi: 10.1016/j.neuron.2024.06.002

Figure Lengend Snippet: (A) Cartoon of U2OS model harboring pB-SNCA-3K-sfGFP transgene. Micrographs show transgene GFP signal in doxycycline-treated cells. (B) Genome-wide CRISPR-Cas9 knockout screen for modifiers of αS toxicity. (C) Volcano plot showing depletion of essential genes (blue). (D) Volcano plot comparing fold-change differential between SNCA-3K-sfGFP and SNCA-WT-sfGFP genotypes. (E) Overlap between spatial (APEX2 and MYTH) and genetic (CRISPR) screen hits. (F) Interaction of actin cytoskeleton-related proteins RhoA and RhoBTB3 with αS by MYTH. (G) Left, Kaplan-Meier curve of single-cell survival tracking in pi-N 3K-sfGFP-pB and pi-N sfGFP-pB models transduced with shRNA lentivirus. Log rank test: *** p < 0.001, **** p < 0.0001. Data are representative of 2 neuronal differentiations with shRNA lentivirus at MOI20. Right, neurite measurement based on RFP signal. (H) PLA of RhoA-pS129 in inclusionopathy models. Bottom, quantification of pS129(+) soma-type inclusions from 3 to 4 independent replicates across 3 separate neuronal differentiations. One-way ANOVA plus Tukey’s multiple comparison test: **** p < 0.0001; n.s., not significant. (I) IF for RhoA, p62, and αS in A53T ( n = 2), E46K ( n = 2), and sporadic ( n = 11) PD brain. Orange arrow, αS(+)/p62(+)/RhoA(+) inclusion; white arrow, αS(+)/p62(+)/RhoA(−) inclusion. (J) Left, quantification of pS129(+)/RhoA(+) inclusions in PD brain. Right, quantification of (I). Error bars = SD.

Article Snippet: We developed algorithms for single-cell inclusion survival tracking with longitudinal imaging (BioStation CT, Nikon) for both spontaneous and seeded inclusionopathy models ( – ).

Techniques: Genome Wide, CRISPR, Knock-Out, Transduction, shRNA, Comparison