Fig 2: Extensive accumulation of ubiquitin-positive aggregates in the spinal cord from SOD1H46R-expressing mice at end-stage. a-c Representative images of double immunostaining with MAP2 (green; neuron marker) and Ubiquitin (red) (a), Ubiquitin (green) and GFAP (red; astrocyte marker) (b), Ubiquitin (green) and Iba1 (red; microglia marker) (c) in the lumbar spinal cord (L4–5) from wild-type (WT) and SQSTM1 (SQSTM1) at 28 weeks of age, SOD1H46R (H46R) and SQSTM1;SOD1H46R (SQSTM1;H46R) mice at end-stage. The nuclei were counterstained with DAPI (blue). Scale bars = 50 μm. b Arrows indicate colocalization of ubiquitin-positive aggregates with GFAP-positive astrocytes. c Arrows indicate colocalization of ubiquitin-positive aggregates with Iba1-positive microglia

Fig 3: Role of NBR1 in adaptive thermogenesis in primary brown adipocytes.a qPCR analysis of Ucp1 expression in primary brown adipocytes differentiated from SVF of four indicated genotypes (n = 5 biological replicates). b Immunoblot analysis of UCP1 in primary brown adipocytes of four indicated genotypes, treated with ISO (0.5 µM) for indicated time. Representative blots (left) and quantification (right) from three independent experiments were shown. c, d Immortalized SVF from WT, Sqstm1–/–, and Sqstm1–/–Nbr1–/– mice were differentiated into brown adipocytes for Seahorse analyses. c Oxygen consumption rate (OCR) were determined (n = 6 biological replicates). Left: plot of OCR to time measured by Seahorse. Right: calculated ISO-stimulated respiration levels after subtracting levels of basal proton leak. d Extracellular acidification rate (ECAR) were measured (n = 6 biological replicates). Left: plot of ECAR to time measured by Seahorse. Right: calculated ISO-stimulated ECAR values after subtracting oligomycin-treated levels. Data are presented as mean ± SEM (a–d). *p < 0.05, **/##p < 0.01, ***p < 0.001, ****/####p < 0.0001. Two-tailed Student’s T-test (a, c, d bar graphs). Two-way ANOVA (b). Source data are provided as a Source Data file.

Fig 4: Quantitative analysis of motor neurons in the anterior horn of lumbar spinal cord. a Representative images for fluorescence Nissl staining in the anterior horn. Nissl positive neurons were detected in the anterior horn of lumbar spinal cord (L4–5) from the mice with four different genotypes; wild-type (WT), SQSTM1, SOD1H46R (H46R), and SQSTM1;SOD1H46R (SQSTM1;H46R) at 16 and 22 weeks of age (wk), and at end-stage (H46R and SQSTM1;H46R) or at 28 wk. (WT and SQSTM1). Cells covering the area of over 240 square pixels (px), corresponding to large motor neurons, are surrounded by red circles, and counted. Scale bars = 50 μm. b Number of large Nissl-positive neurons in the anterior horn of lumbar spinal cord. Vertical axis represents the cumulative number of motor neurons identified in 5 representative Nissl staining images of every tenth serial sections from each sample (n = 3–4, SOD1H46R and SQSTM1;SOD1H46R mice at 22 wk. and end-stage; n = 4, others; n = 3). Values are mean ± s.e.m.. Statistical significance was evaluated by two-way ANOVA with Bonferroni’s post hoc test (*p < 0.05, **p < 0.01)

Fig 5: Overexpression of SQSTM1 increases misfolded SOD1 in the spinal cord of SOD1H46R-expressing mice. a Western blot analysis of SOD1. b Western blot analysis of misfolded SOD1. a, b The spinal cord samples were obtained from wild-type (WT), SQSTM1 (SQSTM1), SOD1H46R (H46R), and SQSTM1;SOD1H46R (SQSTM1;H46R) mice at 16 weeks of age (wk), 22 wk., and end-stage (H46R and SQSTM1;H46R) or 28 wk. (WT and SQSTM1). Two fractions; 1% Triton X-100 soluble and 1% Triton X-100 insoluble/5%SDS soluble fractions were analyzed. SOD1_mono and SOD1_HMW represent monomeric and high molecular-weight forms of SOD1, respectively. SOD1(C4F6) represents misfolded SOD1. GAPDH and β-actin (Actin) were used for a loading control in Triton X-100-soluble and -insoluble fractions, respectively. c Quantification of 1% Triton X-100 soluble SOD1, insoluble SOD1_mono, insoluble SOD1_HMW, soluble misfolded SOD1, and insoluble misfolded SOD1. Values are mean ± s.e.m. (n = 4) in an arbitrary unit relative to H46R mice at 16 weeks of age. Signal intensities were normalized by the levels of GAPDH (soluble fractions) and Actin (insoluble fractions). Statistical significance was evaluated by two-way ANOVA with Bonferroni’s post hoc test (comparisons between different genotypes in the same age; *p < 0.05, **p < 0.01)