Figure 6

NEAT1_2 lncRNA aggregates around the IGAZ forming paraspeckles in motor neurons of the ALS spinal cord. A. Semi-thin sections (0.5 μm thickness) were observed with toluidine blue staining to identify a motor neuron (arrow), which characteristically includes lipofuscin (arrowhead) in the cytoplasm. Solid line, outline of the nucleus; dotted line, outline of the cytoplasm. Scale bar, 50 μm. B. Electron microscopic observation after in situ hybridization using DIG-labeled RNA probe targeting NEAT1_2 lncRNA in the nucleus of a motor neuron in the spinal cord of an ALS patient (Pt B). The interchromatin granule-associated zone (IGAZ) or IGAZ-like zone is observed as an intermediate electron-dense cluster. The gold-labeled probe visualizes NEAT1_2 lncRNA as a halo-shaped pattern of aggregations around IGAZ in the nucleus of an ALS spinal motor neuron (a, b). A control case (C3) demonstrated few NEAT1_2 granules around the IGAZ and/or IGAZ-like zone (c, d). Observation in HeLa cells is shown as a positive control for the formation of paraspeckle structures (e, f). Scale bars, 200 nm (a-d, f) and 500 nm (e).