Progressive loss of dopaminergic neurons in the ventral midbrain of adult mice heterozygote for Engrailed1

Abstract

Engrailed1 and Engrailed2 (En1 and En2) are two developmental genes of the homeogene family expressed in the developing midbrain. En1 and, to a lesser degree, En2 also are expressed in the adult substantia nigra (SN) and ventral tegmental area (VTA), two dopaminergic (DA) nuclei of the ventral midbrain. In an effort to study En1/2 adult functions, we have analyzed the phenotype of mice lacking one En1 allele in an En2 wild-type context. We show that in this mutant the number of DA neurons decreases slowly between 8 and 24 weeks after birth to reach a stable 38 and 23% reduction in the SN and VTA, respectively, and that neuronal loss can be antagonized by En2 recombinant protein infusions in the midbrain. These loss and gain of function experiments firmly establish that En1/2 is a true survival factor for DA neurons in vivo. Neuronal death in the mutant is paralleled by a 37% decrease in striatal DA, with no change in serotonin content. Using established protocols, we show that, compared with their wild-type littermates, En1+/- mice have impaired motor skills, an anhedonic-like behavior, and an enhanced resignation phenotype; they perform poorly in social interactions. However, these mice do not differ from their wild-type littermates in anxiety-measuring tests. Together, these results demonstrate that En1/2 genes have important adult physiological functions. They also suggest that mice lacking only one En1 allele could provide a novel model for the study of diseases associated with progressive DA cell death.

Figure 1.

Progressive DA cell loss in the ventral midbrain of En1+/− mice. A, TH-positive cells in the ventral midbrain of WT and En1+/− mice at 3 and 48 weeks postnatal. B, Progressive loss of DA neurons in SN with age in En1+/− mice (3, 8, 16, 24, 48 weeks postnatal). En1+/− versus WT, **p < 0.01 and ***p < 0.001 (3 vs 8 weeks, ^#p < 0.05; vs 16 weeks, ^##p < 0.01; vs 24 and 48 weeks, ^###p < 0.001). Actual numbers from which percentages of surviving neurons were calculated at each age are provided in supplemental Table 1 (available at www.jneurosci.org as supplemental material). C, Loss of DA neurons in VTA of En1+/− mice (3 and 48 weeks postnatal). For 48 versus 3 weeks, ^#p < 0.05; En1+/− versus WT, **p < 0.01 (n = 3–4 by group of age and genotype).

Figure 2.

Neuronal cells in SN. A, Middle section of the SN (mid-SN) stained for TH. The surface of the SN is outlined by the dashed line. B, Measuring mid-SN surfaces indicates that En1+/− (n = 3) and WT (n = 3) mice show no differences in SN size at 3 and 24 weeks of age. C, TH (red) and NeuN (green) staining of SN cells. Shown are examples of non-DA neurons (arrows) and of DA neurons (arrowheads; yellow; merged picture). D, Decrease of total neuron number in En1+/− versus WT mice between 3 and 48 weeks of age (48 vs 3 weeks, ^##p < 0.01; En1+/− vs WT, ***p < 0.001; n = 3–4 by group of age and genotype). Actual numbers from which percentages of surviving neurons were calculated are provided in supplemental Table 1 (available at www.jneurosci.org as supplemental material). E, Striatal DA contents in WT (n = 8) and En1+/− (n = 10) mice at 55 weeks postnatal (**p < 0.01). F, DA turnover of WT and En1+/− mice at 55 weeks postnatal (***p < 0.001). G, Striatal serotonin (5-HT) contents in WT and En1+/− mice at 55 weeks postnatal (NS).

Figure 3.

Gain of function by intraparenchymal infusion of En2 protein. A, After 14 d of infusion, En2 is detected in a large amount in a region encompassing the SN. Anterior sections are to the left and posterior to the right. B, Progressive loss of DA neurons in SN of En1+/− mice compared with WT expressed as the mean ± SD. At 3 weeks postnatal, the WT and En1+/− mice possess the same number of TH-positive cells in the SN. A decrease of ∼20% is observed at 8 weeks postnatal and of ∼28% at 16 weeks. Between 6 and 9 weeks postnatal, the En1+/− mice lose ∼10% of DA cells. C, Rescue of DA cell loss in En1+/− mice by En2 infusion above the SN. At 9 weeks postnatal, the control En1+/− mice have lost ∼20% of DA cells as compared with the WT (***p < 0.001). No significant difference in TH-positive cell numbers is found between WT infused with saline or WT infused with En2 (p = 0.4; NS). Infusion of En2 in En1+/− mice prevents cell loss in the SN. At 9 weeks postnatal, after 2 weeks of En2 infusion, the number of TH-positive neurons is increased significantly by ∼15% in En1+/− mice as compared with control (**p < 0.01) and is equivalent to the number of DA cells in WT infused with saline or En2.

Figure 4.

Locomotor activity. Performances in an open field of 24-week-old En1+/− and WT mice expressed as the mean ± SD of distance traveled in centimeters (A), number (mean ± SD) of rearings (B), and time spent in the central zone (C); **p < 0.01. D, Effect of amphetamine (2 mg/kg) on the distance traveled in the open field (in centimeters; mean ± SD) by WT and En1+/− mice during the 30 min that follow injection. Mice are 32–33 weeks old at the time of the test. Animals receive either saline (Sal) or amphetamine (Amph). Differences (Student's t test): En1+/− versus WT, *p < 0.05; Amph versus Sal, ^#p < 0.05 and ^##p < 0.01. Amphetamine administration at a dose of 5 mg/kg induces hyperactivity in both WT and En1+/− mice (data not shown). E, Rotarod performances of 27-week-old En1+/− and WT mice expressed as the mean ± SD of latency (s) to fall (average of 3 trials). Although the same trend (decreased performance for En1+/− mice) was observed at all speeds, the difference did not reach significance because of the abnormally high performances of one mutant mouse of 10 (see Results).

Figure 5.

Forced swimming test. Performances of 26-week-old En1+/− and WT mice in forced swimming test are expressed as the mean ± SD of immobility time during the first (A) and the second (B) test exposure; *p < 0.05 and **p < 0.01. C, Performances in an open field of 24-week-old En1+/− and WT mice tested in FST expressed as the mean ± SD of the distance traveled (in centimeters); *p < 0.05.

Figure 6.

Saccharin consumption test. Shown is the Preference Index for saccharin (A) and total fluid intake (B) in 35-week-old En1+/− and WT mice expressed as the mean ± SD during a two-bottle free choice paradigm of saccharin consumption; *p < 0.05 and ***p < 0.001.