Oxidative stress is responsible for deficient survival and dendritogenesis in purkinje neurons from ataxia-telangiectasia mutated mutant mice

Abstract

Atm gene-disrupted mice recapitulate the majority of characteristics observed in patients with the genetic disorder ataxia-telangiectasia (A-T). However, although they exhibit defects in neuromotor function and a distinct neurological phenotype, they do not show the progressive neurodegeneration seen in human patients, but there is evidence that ataxia-telangiectasia mutated (Atm)-deficient animals have elevated levels of oxidized macromolecules and some neuropathology. We report here that in vitro survival of cerebellar Purkinje cells from both Atm "knock-out" and Atm "knock-in" mice was significantly reduced compared with their wild-type littermates. Although most of the Purkinje neurons from wild-type mice exhibited extensive dendritic elongation and branching under these conditions, most neurons from Atm-deficient mice had dramatically reduced dendritic branching. An antioxidant (isoindoline nitroxide) prevented Purkinje cell death in Atm-deficient mice and enhanced dendritogenesis to wild-type levels. Furthermore, administration of the antioxidant throughout pregnancy had a small enhancing effect on Purkinje neuron survival in Atm gene-disrupted animals and protected against oxidative stress in older animals. These data provide strong evidence for a defect in the cerebellum of Atm-deficient mice and suggest that oxidative stress contributes to this phenotype.

Figure 1.

Structure of the isoindoline nitroxide, CTMIO. This compound was synthesized as described by Bottle et al. (2000) and is water soluble up to 2 mm.

Figure 2.

Survival and differentiation of Purkinje cells in culture. A, Calbindin-stained Purkinje cells from wild-type and Atm-Δ SRI homozygous mice. a-c are typical examples of dentritogenesis in wild-type (+/+) Purkinje neurons, and d-f are corresponding neurons from Atm-ΔSRI cultures (Atm^ΔSRI/ΔSRI). B, Calbindin-stained Purkinje neurons from Atm^-/- mice. a-f are typical examples of Purkinje cells from Atm^-/--deficient mice. Note the poor degree of differentiation compared with wild-type cells. All Purkinje neurons were isolated from cerebella of day 0 mice and maintained in culture for 10 d as described in Materials and Methods.

Figure 3.

Effect of the nitroxide CTMIO on dentritogenesis in Purkinje neurons isolated from the cerebella of Atm-Δ SRI homozygous mice (Atm^ΔSRI/ΔSRI). Neurons from Atm^ΔSRI/ΔSRI mice were plated out at day 0 in the absence (a, b) or presence (c, d) of CTMIO (100 μm). Additional CTMIO (100 μm) was added to the cultures at 3 d intervals for the 10 d period of culture. Samples were fixed and calbindin staining was performed to detect surviving Purkinje cells. The neurons depicted are representative of the extent of dentritogenesis observed in treated (c, d) and untreated (a, b) cultures.

Figure 4.

Immunostaining of Purkinje cells from wild-type, Atm^{- / -}, and Atm-^ΔSRI/ΔSRI homozygous mice cerebella sections for 3-nitrotyrosine. Cerebella were isolated from 2-month-old mice treated or untreated with CTMIO antioxidant and fixed in paraformaldehyde; 5 μm sections were prepared and incubated with anti-nitrotyrosine antibody and HRP-linked secondary antibody. CTMIO was administered intraperitoneally to pregnant female mice at 3 d intervals throughout pregnancy and during weaning. Thereafter CTMIO (100 μm) was added to the drinking water.