Abnormal development of Purkinje cells and lymphocytes in Atm mutant mice

Abstract

Motor incoordination, immune deficiencies, and an increased risk of cancer are the characteristic features of the hereditary disease ataxia-telangiectasia (A-T), which is caused by mutations in the ATM gene. Through gene targeting, we have generated a line of Atm mutant mice, Atm(y/y) mice. In contrast to other Atm mutant mice, Atm(y/y) mice show a lower incidence of thymic lymphoma and survive beyond a few months of age. Atm(y/y) mice exhibit deficits in motor learning indicative of cerebellar dysfunction. Even though we found no gross cerebellar degeneration in older Atm(y/y) animals, ectopic and abnormally differentiated Purkinje cells were apparent in mutant mice of all ages. These findings establish that some neuropathological abnormalities seen in A-T patients also are present in Atm mutant mice. In addition, we report a previously unrecognized effect of Atm deficiency on development or maintenance of CD4(+)8(+) thymocytes. We discuss these findings in the context of the hypothesis that abnormal development of Purkinje cells and lymphocytes contributes to the pathogenesis of A-T.

Figure 1

Generation of Atm^y/y mice and Atm expression. (A) Location of the Atm^y mutation and other reported murine Atm mutations; a (14), b (15, 16), and c (13). B, BamHI; H, HindIII; Bg, BglII; Sc, SacI; Xb, XbaI; and Neo^r, neomycin-resistance gene. (B) Southern blot analysis of Atm^y mutation. BamHI-digested DNA is probed with the indicated 3′ probe (A). (C) Northern blot analysis of Atm expression. Total RNA from brain, lymph node, and thymus of wild-type (+/+) and Atm^y/y (y/y) mice were probed with an Atm cDNA fragment (Lower) and reprobed with a DNA-PKcs cDNA probe (Upper) to confirm equal RNA loading. (D and E) Western blot analysis of Atm expression in Atm^y/y tissues using antibodies raised against the C terminus (D) and N terminus (E) of the ATM protein. The bands at ≈200 kDa and ≈300 kDa are likely nonspecific since they are present in human and murine samples of both genotypes. (F) Kaplan–Meier survival curve of Atm^y/y mice (n = 55).

Figure 2

Abnormal T lymphocyte development in Atm^y/y mice. (A) Average pairwise comparisons of weight, total numbers of thymocytes, and splenocytes from 10 same-sex littermates pairs 1–5 mo old. (B) (Upper) The average reduction of the total number of each Atm^y/y thymocyte subpopulations (DN, CD4⁻8⁻; DP; CD4⁺8⁺; CD4⁺SP, CD4⁺8⁻; CD8⁺SP, CD4⁻8⁺) from seven littermate pairs. (Lower) FACS analysis of T cell development in Atm^y/y thymuses. (C) Histogram of CD3 surface expression in Atm^y/y thymocytes (y/y). (Lower) FSC (forward site scatter) using a lymphocyte gate on CD3^low and CD3^int DP cells, respectively. (D) Splenic T cells in Atm^y/y mice (y/y) were reduced in numbers, but their surface phenotype (TCRβ/CD3 expression) appeared to be normal. Numbers indicate the percentage of splenic T cells (CD3⁺) and B cells (B220⁺).

Figure 3

Motor-learning deficits and dendritic changes in Atm^y/y mice. (A) Performance of wild-type mice but not Atm^y/y mice on an accelerating rotating rod improved over the trial period (P < 0.01 by repeated-measure ANOVA, regression slope = 1.21 s/day, P < 0.05), and by the seventh trial day, wild-type mice performed better than Atm^y/y mice (P < 0.02, Mann–Whitney rank sum test). (B) Parvalbumin immunohistochemistry of midsagittal cerebellar sections from 5-mo-old wild-type (Upper) and Atm^y/y mice (Lower) reveals the altered dendritic arborization of Purkinje cells in Atm^y/y mice. (C) This can also be observed in Bodian-stained sections from 1-yr-old mice, wild-type (Upper), and Atm^y/y (Lower). (D) Lucifer yellow-injected wild-type and Atm^y/y Purkinje cells displaying either the prototypic morphology (+/+) or premature dendritic branching (y/y), which can result in multiple dendrites emanating from the soma (upper y/y cell). (E) Cumulative percent graph of stem dendrite length; 7 of 26 Atm^y/y Purkinje cells have multiple dendrites. (Scale bar in B and C is 50 μm.)

Figure 4

(A and B) An ectopic Purkinje cell in an Atm^y/y mouse. Ectopic cells were located and counted by double labeling for calbindin immunoreactivity (A) and propidium iodide staining (B). (C) Varicosity along Purkinje cell axon. (Scale bar in A is 50 μm and in C is 20 μm.)