2B7 reaches spinal cord neurons and induces trophic signals in wild type mice and in ALS mice.
(A) 2B7 was labeled with Atto-488 fluorochrome and 15 μg 2B7-ATTO were injected intraperitoneally into 6-week old C57BL/6J wild type mice. After 48 hrs, mice were perfused, and sections of their spinal cord examined. Staining was observed in ventral motor neurons of lumbar spinal cord (smaller pictures showing only one layer), and in confocal images (large picture on left). The label within subcellular organelles can be detected, indicative of internalized fluorochrome within vesicles. Motor neurons at all levels of the spinal cord contained the label. (B) A single intraperitoneal injection of 2B7 was done in wild type C57BL/6J mice (n = 3 per group), spinal cords were collected after 18 hrs, and detergent lysates of soft tissues were prepared and studied by Western blots. Quantification of p-Akt or p-PLCγ was done by densitometric analysis versus actin to standardize loading. The data shows activated p-Akt, and p-PLCγ in 2B7–treated mice, when compared to control IgG–treated mice. t-test * p<0.05 versus control. (C) The quantitative data in (B) was validated using symptomatic ALS mice (post-natal day 102). ALS mice received a single intraperitoneal injection of 2B7 or non-binding control IgG (n = 5 per group). After ~18 hrs spinal cords were collected for biochemical and immunohistochemical studies. Detergent lysates of soft tissues were studied by Western blots using with anti-p-Akt, anti-p-MAPK, or anti-actin to standardize loading. The data suggests activated p-Akt, and p-MAPK in 2B7–treated ALS mice, when compared to control IgG–treated ALS mice. t-test * p<0.05 and ** p<0.01 versus control. (D) Immunohistochemical studies of ALS spinal cords. Cryosections of 2B7-treated or control-treated ALS mice (as in panel C) were immunostained with anti-p-Akt and anti-p-MAPK antibodies. Fluorescent microscopy data shows that 2B7-treatment induced Akt activation in neuronal cell bodies in the gray matter (co-localized with the NeuN marker) and in neuronal fibers of the white matter; and MAPK activation almost exclusively in neuronal cell bodies, but not in neuronal fibers of the white matter. Akt and MAPK activation in neurons are not seen in control IgG-treated ALS samples.