Age-dependent maintenance of motor control and corticostriatal innervation by death receptor 3

Abstract

Death receptor 3 is a proinflammatory member of the immunomodulatory tumor necrosis factor receptor superfamily, which has been implicated in several inflammatory diseases such as arthritis and inflammatory bowel disease. Intriguingly however, constitutive DR3 expression has been detected in the brains of mice, rats, and humans, although its neurological function remains unknown. By mapping the normal brain expression pattern of DR3, we found that DR3 is expressed specifically by cells of the neuron lineage in a developmentally regulated and region-specific pattern. Behavioral studies on DR3-deficient (DR3(ko)) mice showed that constitutive neuronal DR3 expression was required for stable motor control function in the aging adult. DR3(ko) mice progressively developed behavioral defects characterized by altered gait, dyskinesia, and hyperactivity, which were associated with elevated dopamine and lower serotonin levels in the striatum. Importantly, retrograde tracing showed that absence of DR3 expression led to the loss of corticostriatal innervation without significant neuronal loss in aged DR3(ko) mice. These studies indicate that DR3 plays a key nonredundant role in the retention of normal motor control function during aging in mice and implicate DR3 in progressive neurological disease.

Figure 1.

Developmental DR3 expression in the early postnatal murine brain. Shown is DR3 promoter-driven β-Gal expression (X-Gal, blue) in the brains of early postnatal DR3^ko mice. Low magnification is shown in left-hand panels; high magnification in the right-hand panels at postnatal days 1 to 9 (P1 to P9) as indicated. Scale bars: 500 μm for left-hand panels; 100 μm for right-hand panels. The dentate gyrus is shown in each case with images representative of 2 animals per age.

Figure 2.

DR3 and TL1A expression in the adult brain. A, B, Representative images of β-Gal staining in adult DR3^ko and DR3^het mice in different areas of the brain. Number of β-Gal-stained cells increased in the cortex of DR3^ko mice, particularly in prefrontal areas such as the anterior olfactory nuclei (Ai, Aii) and generally across other areas such as the primary motor cortex (Biv, Bv), but remained high and unchanged in the dentate gyrus (Bvi, Bvii). Comparative expression is shown for the prefrontal (Aiii) and motor cortex (Bviii). Areas in which β-Gal expression was detected are shown for DR3^ko in yellow and DR3^het in blue. Filled areas show high-density β-Gal staining. Areas from which high-magnification sections are shown (Ai, Aii, Biv–Bvii) are indicated by numbers with red squares for DR3^ko and green squares for DR3^het animals. C, DR3 expression in DR3^het brain as detected using the β-Gal reporter. BG, Basal Ganglia; Co, cortex; Cb, cerebellum; DG, dentate gyrus; Hf, hippocampal formation. D, Expression of DR3 and TL1A mRNA in the indicated areas of the brain as measured by RT-PCR. GAPDH is shown as a loading control. E, Microscopy showing neuron-specific DR3 promoter-driven β-Gal expression in the dentate gyrus using immunofluoromicroscopy. Signals from β-Gal overlap with NeuN (Ei–Eiii), but not GFAP (Eiv–Evi).

Figure 3.

Time course of development of observable gait disorders in DR3^ko mice. Proportion of DR3^ko and DR3^het mice showing observable gait disorder with age. Numbers under the graph represent numbers of mice per age group assessed.

Figure 4.

Defects in gait in aged DR3^ko mice. Gait defects in 3-month- or 2-year-old mice were compared using timed footprint and movement analysis. A, Average run time for young and old mice. B, Representative footprints of DR3^ko and DR3^het mice are shown. C, Footprint analysis showing stride length (i, ii) and stride width and overlap (iii, iv) of young (i, iii) and old (ii, iv) mice. *Student's t tests showed significant differences as indicated.

Figure 5.

Defects in balance in aged DR3^ko mice. Balance was compared in 2-year-old DR3^het and DR3^ko mice by timing of runs along a raised balance beam within a 60 cm run. A, Completion frequency for DR3^het and DR3^ko mice. χ² test showed significant difference as indicated. B, Average run times for DR3^het and DR3^ko mice, with animals not completing given a maximum time of 120 s. Each point represents the average from 3 runs from an individual animal. *Student's t test assuming unequal variance showed significant differences as indicated. C, Representative footprints of DR3^ko and DR3^het mice are shown.

Figure 6.

Hyperkinesia and increased movement complexity in DR3^ko mice. A, Representative plots of the movement of digitally tracked mice in a 1 m square open field over 1 min (n = 6–8 per group). S, Start; F, finish. B, Accumulative distance traversed (i, ii) and accumulative frequency of path-crossing events (iii, iv) displayed by young (i, iii) and old (ii, iv) DR3^ko and DR3^het mice during 10 min. C, Average distance traversed before mice crossed their own paths. Each group included 4–8 mice, and data are representative of four experiments. Asterisks in relevant panels indicate significance of Student's t tests. Lines and error bars represent mean ± SEM.

Figure 7.

Expression of tyrosine hydroxylase in the brain. A, Immunohistochemistry showing TH expression in DR3^ko (i) and DR3^wt (ii) mice. B, Summary data showing no difference in TH expression at all matched levels throughout the striatum between DR3^ko and DR3^wt mice.

Figure 8.

Alterations in corticostriatal innervation in the absence of changes of neuronal number in DR3^ko mice. A, Stereological counting of NeuN⁺ cells showed no difference between DR3^ko and DR3^het mice at 4 different matched levels in the brain. Data are mean ± SEM from n = 9 mice. B, C, Retrograde tracing of innervation into the striatum. B, Representative images from the cortex of 18-month-old DR3^ko (i) and DR3^wt (ii) mice. Scale bars, 100 μm. C, Young (3 months of age) DR3^ko and DR3^wt mice showed equivalent corticostriatal innervation, while old (18 months of age) DR3^ko mice showed a reduction in corticostriatal innervation compared to age-matched DR3^wt mice. Data are percentage of the age-matched DR3^wt mean ± SEM from n = 4 mice. Statistical significance of *p < 0.03 using a Student's t test.