Figure 4

Netrin-1 regulates net Aβ peptide level. (a) Brain slices from PDAPP transgenic mice and control nontransgenic littermates (N-PDAPP) were cultured in the presence or absence of netrin-1 (1.5 nM). Supernatants were harvested after 3–5 days and evaluated by ELISA assay for Aβ1–40 and Aβ1–42. In all, 90 ng/ml of netrin-1 were added to all Aβ standards to rule out netrin-1 interference with binding of the antibodies used in the ELISA to their epitopes on Aβ. NGF (250 ng/ml) or IGF-1 (100 ng/ml) was also added as control and failed to have any effect on Aβ level (not shown). (b) Netrin-1 expression (inset) and net Aβ production were measured in 5–7-month old PDAPP/netrin-1+/− or PDAPP/netrin-1+/+ mice by ELISA. Fold increase is presented as the ratio between average Aβ levels detected in PDAPP/netrin-1+/− mice and that in PDAPP/netrin-1+/+ mice. Four cohorts of the animals of similar age (netrin-1 +/+ and +/−) were studied. Total number of mice studied were 16. ANOVA test was used comparing +/− versus +/+ in the four groups (P<0.027), comparing +/− versus +/+ in the whole population (P=0.0005). (c) Netrin-1 immunoblot in mouse nervous system. Cortex (Ctx), cerebellum (Cb), spinal cord (ASC), striatum (Str), and hippocampus (Hi) from adult mice or as control E13 mice embryonic spinal cord (ESC) were dissected out, and immunoblots using antinetrin-1 or anti-β-actin (as a loading control) antibody are shown. (d) Netrin-1 expression in adult brain followed by LacZ activity in netrin-1 +/− mutant mice. Control: Xgal staining on net+/+ brain mouse. Cortex (Ctx), cerebellum (Cb), striatum (Str), and hippocampus (Hi) are indicated. Netrin-1 transcript in adult brain was followed using the analysis of transgenic mouse in which the netrin-1 promoter drives the expression of LacZ.⁹ Brains of 8 months old netrin-1 mutant mice net +/− or net +/+ in the NPDAPP genetic background were excised and incubated overnight with a 1.3 mg/ml X-Gal solution, then fixed for 30 min at room temperature with PFA 4% and included in 3% low melting agarose (Cambrex). Brain sections (200 µm) were performed using a vibratome and incubated for 2 days with 1.3 mg/ml X-Gal solution (Euromedex) at 4 °C. (e and f) 6–8 months PDAPP transgenic mice (n=16) were infused intracerebrally with 100 µl of artificial cerebrospinal fluid (saline) with or without 130 µg/ml recombinant mouse netrin-1 for 12–13 days using Alzet osmotic pumps. (e) Aβ–40 and Aβ1–42 levels measured by ELISA as in panel b. (f) preference for novel stimuli in the ON test as described in the Materials and Methods section