Fig. 1

Paraspeckles are formed in the spinal cord of sALS and fALS patients but not healthy controls. a and b Examples of spinal motor neurons with paraspeckles (a) and their quantification (b) in ALS patients with different disease aetiology. Paraspeckles were visualised in the spinal cord sections of a cohort of fALS and sALS patients as well as neurologically normal control individuals using RNA-FISH with a fluorescent (Quasar 570) probe mapping to the 5′ portion of NEAT1. Images were also taken in the FITC channel to distinguish between specific NEAT1 signal and green autofluorescence from lipofuscin (a). The fraction of neurons with identifiable paraspeckles in the spinal anterior horn of aetiologically different ALS cases and control individuals was quantified and plotted separately for fALS with TARDBP mutations (ALS-TDP), fALS with C9ORF72 repeat expansion (ALS-C9) and sALS cases (b). The top figure within each bar corresponds to the number of cases analysed and the figures below - to the number of individual neurons negative or positive for the presence of paraspeckles. Scale bars, 10 μm. c Examples of paraspeckle-containing neurons in the ALS spinal cord visualised with RNAscope® NEAT1_2 specific probe. In the bottom panel, a paraspeckle-positive (right) and a paraspeckle-negative (left) neurons, found adjacent to each other, are shown. Nuclei are circled. Scale bar, 10 μm. d NEAT1 levels in the total RNA samples extracted from transversely cut spinal cord blocks of ALS patients and healthy controls analysed by qRT-PCR (n = 4 for control and ALS patients, including two sALS and two ALS-C9 cases, Mann-Whitney U-test). e NEAT1_2 levels in neurons microdissected from the spinal anterior horn of ALS patients and healthy controls analysed by qRT-PCR (n = 3 for control and n = 6 for ALS cases, including three sALS and three ALS-C9 cases, Mann-Whitney U-test). f Paraspeckles in glial cells in the ALS spinal cord visualised with RNAscope® ISH using NEAT1_2 specific probe. Representative images of the spinal cord for a control individual and an ALS patient are shown. Scale bar, 20 μm