Fluorescence microscopic tools are widely used for assessing mitochondrial biology, however quantitative assays of parameters such as organelle dynamics are not trivial. High‐content imaging systems established the need for scalable assays that are robust and are able to evaluate biological parameters in an automated, unsupervised manner. We introduce here a software platform implementing novel image processing techniques allowing low light level but robust determination of mitochondrial swelling and organelle motion in cultured cells. Mitochondrial swelling is measured by the ratio of fluorescence intensities in high over low frequency spatial band pass filtered copies of the same image. This ratio is highly sensitive to mitochondrial swelling, but insensitive to fission or fusion, motion and overlaps of mitochondria. Organelle transport is assayed by optical flow, featuring instantaneous velocity determination from a pair of images by detecting motion of edges. Optical flow provides velocity vectors; therefore anterograde, retrograde transport and local, wiggling motion can be distinguished. Both assays are scalable, applicable for imaging in microplates and are accessible through a user friendly interface.
Abstract Exposure of neurones in culture to excitotoxic levels of glutamate results in an initial transient spike in [Ca 2+ ] i followed by a delayed, irreversible [Ca 2+ ] i rise governed by rapid kinetics, with Ca 2+ originating from the extracellular medium. The molecular mechanism responsible for the secondary Ca 2+ rise is unknown. Here, we report that the delayed Ca 2+ entry in cortical neurones is diminished by 2‐aminoethoxydiphenyl borate (2‐APB: IC 50 = 62 ± 9 µ m ) and La 3+ (IC 50 = 7.2 ± 3 µ m ), both known to inhibit transient receptor potential (TRP) and store‐operated Ca 2+ (SOC) channels. Application of thapsigargin, however, failed to exacerbate the delayed Ca 2+ deregulation, arguing against a store depletion event as the stimulus for induction of the secondary [Ca 2+ ] i rise. In addition, these neurones did not exhibit SOC entry. Unexpectedly, application of ryanodine or caffeine significantly inhibited glutamate‐induced delayed Ca 2+ deregulation. In basal Ca 2+ entry experiments, La 3+ and 2‐APB modulated the rapid rise in [Ca 2+ ] i caused by exposure of neurones to Ca 2+ after pre‐incubating in a calcium‐free medium. This basal Ca 2+ influx was mitigated by extracellular Mg 2+ but not aggravated by thapsigargin, ryanodine or caffeine. These results indicate that 2‐APB and La 3+ influence non‐store‐operated Ca 2+ influx in cortical neurones and that this route of Ca 2+ entry is involved in glutamate‐induced delayed Ca 2+ deregulation.
Abstract Microgravity is associated with immunological dysfunction, though the mechanisms are poorly understood. Here, using single-cell analysis of human peripheral blood mononuclear cells (PBMCs) exposed to short term (25 hours) simulated microgravity, we characterize altered genes and pathways at basal and stimulated states with a Toll-like Receptor-7/8 agonist. We validate single-cell analysis by RNA sequencing and super-resolution microscopy, and against data from the Inspiration-4 (I4) mission, JAXA (Cell-Free Epigenome) mission, Twins study, and spleens from mice on the International Space Station. Overall, microgravity alters specific pathways for optimal immunity, including the cytoskeleton, interferon signaling, pyroptosis, temperature-shock, innate inflammation (e.g., Coronavirus pathogenesis pathway and IL-6 signaling), nuclear receptors, and sirtuin signaling. Microgravity directs monocyte inflammatory parameters, and impairs T cell and NK cell functionality. Using machine learning, we identify numerous compounds linking microgravity to immune cell transcription, and demonstrate that the flavonol, quercetin, can reverse most abnormal pathways. These results define immune cell alterations in microgravity, and provide opportunities for countermeasures to maintain normal immunity in space.
Cyclophilin D (cypD)-deficient mice exhibit resistance to focal cerebral ischemia and to necrotic but not apoptotic stimuli. To address this disparity, we investigated isolated brain and in situ neuronal and astrocytic mitochondria from cypD-deficient and wild-type mice. Isolated mitochondria were challenged by high Ca(2+), and the effects of substrates and respiratory chain inhibitors were evaluated on permeability transition pore opening by light scatter. In situ neuronal and astrocytic mitochondria were visualized by mito-DsRed2 targeting and challenged by calcimycin, and the effects of glucose, NaCN, and an uncoupler were evaluated by measuring mitochondrial volume. In isolated mitochondria, Ca(2+) caused a large cypD-dependent change in light scatter in the absence of substrates that was insensitive to Ruthenium red or Ru360. Uniporter inhibitors only partially affected the entry of free Ca(2+) in the matrix. Inhibition of complex III/IV negated the effect of substrates, but inhibition of complex I was protective. Mitochondria within neurons and astrocytes exhibited cypD-independent swelling that was dramatically hastened when NaCN and 2-deoxyglucose were present in a glucose-free medium during calcimycin treatment. In the presence of an uncoupler, cypD-deficient astrocytic mitochondria performed better than wild-type mitochondria, whereas the opposite was observed in neurons. Neuronal mitochondria were examined further during glutamate-induced delayed Ca(2+) deregulation. CypD-knock-out mitochondria exhibited an absence or a delay in the onset of mitochondrial swelling after glutamate application. Apparently, some conditions involving deenergization render cypD an important modulator of PTP in the brain. These findings could explain why absence of cypD protects against necrotic (deenergized mitochondria), but not apoptotic (energized mitochondria) stimuli.
X-linked dystonia-parkinsonism (XDP) is a rare neurodegenerative disease endemic to the Philippines. The genetic cause for XDP is an insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within intron 32 of TATA-binding protein associated factor 1 (TAF1) that causes an alteration of TAF1 splicing, partial intron retention, and decreased transcription. Although TAF1 is expressed in all organs, medium spiny neurons (MSNs) within the striatum are one of the cell types most affected in XDP. To define how mutations in the TAF1 gene lead to MSN vulnerability, we carried out a proteomic analysis of human XDP patient-derived neural stem cells (NSCs) and MSNs derived from induced pluripotent stem cells. NSCs and MSNs were grown in parallel and subjected to quantitative proteomic analysis in data-independent acquisition mode on the Orbitrap Eclipse Tribrid mass spectrometer. Subsequent functional enrichment analysis demonstrated that neurodegenerative disease-related pathways, such as Huntington's disease, spinocerebellar ataxia, cellular senescence, mitochondrial function and RNA binding metabolism, were highly represented. We used weighted coexpression network analysis (WGCNA) of the NSC and MSN proteomic data set to uncover disease-driving network modules. Three of the modules significantly correlated with XDP genotype when compared to the non-affected control and were enriched for DNA helicase and nuclear chromatin assembly, mitochondrial disassembly, RNA location and mRNA processing. Consistent with aberrant mRNA processing, we found splicing and intron retention of TAF1 intron 32 in XDP MSN. We also identified TAF1 as one of the top enriched transcription factors, along with YY1, ATF2, USF1 and MYC. Notably, YY1 has been implicated in genetic forms of dystonia. Overall, our proteomic data set constitutes a valuable resource to understand mechanisms relevant to TAF1 dysregulation and to identify new therapeutic targets for XDP.
