Extended Data Fig. 10: Intra-cerebrospinal fluid injection of CHIT1 induces accelerated MN senescence in monkey spinal cords.

a, ELISA analysis of CHIT1 concentration before vehicle- and CHIT1-treated monkey CSF at baseline. b, Imagiological diagnosis to confirm the implantation position of the Ommaya reservoir into the monkey ventricle by using 3D-reconstructed CT image. The arrows indicate the Ommaya reservoir. c, SA-β-Gal staining of the spinal vGM sections from vehicle- or CHIT1-treated monkey spinal cords. Representative images (left) and the percentage of SA-β-Gal-positive areas in the total vGM area (right) are shown. The arrows indicate SA-β-Gal-positive areas. The dashed lines indicate the boundary of vGM. Scale bars, 100 μm. d, The percentage of SA-β-Gal⁺ MNs or SA-β-Gal⁺ non-MN cells in total SA-β-Gal⁺ cells in the spinal vGM of CHIT1-treated monkeys. e, Immunofluorescence staining of 4-HNE in spinal cords from vehicle- or CHIT1-treated monkeys. Representative images (left) and the mean 4-HNE intensity per MN (right) are shown. The arrows indicate the MNs. Scale bars, 50 μm. f, Immunohistochemical staining of ChAT (left) or γ-H2A.X (right) in the adjacent sections from vehicle- or CHIT1-treated monkey spinal cords. Representative images of the vGM region (left) and the percentage of γ-H2A.X⁺ MNs in total MNs (right) are shown. Scale bars, 100 μm. Scale bars (zoomed in), 20 μm. The arrow indicates the γ-H2A.X⁺ MN. g, Representative images of MN morphology in vehicle- or CHIT1-treated monkey spinal cords. Scale bars, 100 μm. h, Sholl analysis of basal and apical dendrites crossing through a series of concentric circles centred at the MN soma and spaced at 15 μm intervals of spinal cords from vehicle- or CHIT1-treated monkeys. All MNs from three discontinuous cross-sectional slices per monkey were detected. i, Golgi staining of vehicle- or CHIT1-treated monkey spinal cords. Representative images (left) and quantification of spine numbers per 10 μm apical dendrites of neurons (right) are shown. More than 80 apical dendrites of MNs from three discontinuous cross-sectional slices per monkey were detected. Scale bars, 5 μm. j, Immunofluorescence staining of VAChT and ChAT in spinal cords from vehicle- or CHIT1-treated monkeys. Representative images (left) and the puncta number per MN (right) are shown. Scale bars, 50 μm. k, Co-staining of BTX and NF in the NMJ of skeletal muscle from vehicle- or CHIT1-treated monkeys. Representative images (left) and the percentage of nerve terminal area per 0.2 cm² or the percentage of axon area per 0.2 cm² (right) are shown. Scale bars, 50 μm. l, Representative evoked field currents in the distal limbs from vehicle- and CHIT1-treated monkeys (left), and the statistics of the amplitude of evoked currents (right) are shown. m, Line graph showing the cumulative change in movement distance in four hours of vehicle- or CHIT1-treated monkeys at baseline. The eight young monkeys were randomly divided into two groups, vehicle- and CHIT1-treated. The statistics data (Extended Data Fig. 10m–p) come from the eight young monkeys (Fig. 1i–k and Extended Data Fig. 2j). n, Bar plot showing movement distance in four hours of vehicle- or CHIT1-treated monkeys at baseline. o, Bar plot showing active time over a four-hour period of vehicle- or CHIT1-treated monkeys at baseline. p, Bar plot showing frequency of food grasping (in number of times) over a five-hour period of vehicle- or CHIT1-treated monkeys at baseline. Data (a, c, e-f, h-p) are shown as the mean ± s.e.m. P values were calculated using two-tailed unpaired t-tests. Three discontinuous cross-sectional slices per monkey (c-f, h-k) and n = 4 monkeys per group (a, c-f, h-k, m-p). n = 3 monkeys per group (l).

Source Data