Journal: Journal of Neurochemistry
Article Title: Intracerebroventricular administration of Cystatin C ameliorates disease in SOD1‐linked amyotrophic lateral sclerosis mice
Figure Lengend Snippet: Cystatin C (CysC) administration reduces insoluble p62 and poly‐ubiquitin accumulation in the lumbar spinal cords of early symptomatic SOD1 G93A mice. (a) Immunoblotting analyses of p62 and poly‐ubiquitin (poly‐Ubi). Note that detergent (1% Triton X‐100)‐insoluble p62 and poly‐Ubi was accumulated in SOD1 G93A mice, but they were not affected by CysC administration. (b) Co‐localization of p62 and poly‐Ubi aggregates were predominantly observed in outside of neurons. A representative image for the lumbar spinal cord section of the end‐stage SOD1 G93A mice with CysC administration stained for p62, poly‐ubiquitin, and CysC, along with a merged image with DAPI. (c) Immunoblotting analyses of spinal cords derived from the early symptomatic stage (4 months old) SOD1 G93A mice administered with phosphate‐buffered saline (PBS) or CysC for 2 weeks. In contrast to the end‐stage samples (a), the amounts of insoluble p62 and poly‐ubiqutinated proteins were reduced by CysC administration, suggesting the improved protein degradation. It should be also noted that bands corresponding to LC3‐II were hardly detected (arrowhead). (d) Astrocytes and microglia in the lumbar spinal cords of end‐stage SOD1 G93A mice, with or without administration of CysC, were immunostained with antibodies against glial fibrillary acidic protein (GFAP) and Iba‐1 with DAPI. Scale bars: 50 μm (b), and 100 μm (d).
Article Snippet: Antibodies used were as follows: anti‐phosphorylated AMPK (Thr172) (1:250, #sc‐33524, RRID: AB_2169714, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti‐LC3 (1:500, #NB100‐2331, RRID: AB_10001955, Novus Biologicals LLC, Littleton, CO, USA), anti‐CysC (1 : 500 for immunoblotting, 1 : 100 for immunohistochemistry, #ab109508, RRID: AB_10888303, Abcam, Cambridge, UK), anti‐β‐actin (1 : 5000, #5441, RRID: AB_476744, Sigma‐Aldrich Co LLC, St. Louis, MO, USA), anti‐influenza hemagglutinin‐tag (HA‐tag) (1 : 500, #11‐867‐423‐001, RRID: AB_10094468, Roche, Basel, Switzerland), anti‐p62 (for immunoblotting, 1 : 1000, #PM045, RRID: AB_2169714, Medical and Biological Laboratories (MBL), Nagoya, Japan) (for immunohistochemistry, 1 : 100, #03‐GP62‐C, RRID: AB_1542690, American Research Products Inc., Belmont, MA, USA), anti‐poly‐ubiquitin (poly‐Ubi) (1 : 1000 for immunoblotting, 1:500 for immunohistochemistry, #D058‐3, RRID: AB_592937, MBL), anti‐glial fibrillary acidic protein (1 : 250, #G3893, RRID: AB_477010, Sigma‐Aldrich), and anti‐Iba‐1 (1:500, #019‐10741, Wako Pure Chemical Industries Ltd., Osaka, Japan).
Techniques: Mouse Assay, Staining, Derivative Assay