Tau haploinsufficiency causes prenatal loss of dopaminergic neurons in the ventral tegmental area and reduction of transcription factor orthodenticle homeobox 2 expression

Abstract

Homozygous tau knockout (Mapt^-/-) mice develop age-dependent dopaminergic (DA) neuronal loss in the substantia nigra (SN) and ventral tegmental area (VTA), supporting an important function of tau in maintaining the survival of midbrain dopaminergic neurons (mDANs) during aging. However, it remains to be determined whether the microtubule-associated protein tau regulates the differentiation and survival of mDANs during embryonic developmental stages. Here, we show that tau haploinsufficiency in postnatal day 0 (P0) heterozygous (Mapt^+/-) pups, but not a complete loss of tau in the Mapt^-/- littermates, led to a significant reduction of DA neurons in the VTA. This selective loss of DA neurons correlated with a similar reduction in orthodenticle homeobox 2 (Otx2), which is restricted to VTA neurons at the postmitotic stage and selectively controls the neurogenesis and survival of specific neuronal subtypes of VTA. Moreover, the prenatal developmental cell death in the Mapt^+/- VTA specifically increased, and the expression of microtubule-associated protein (MAP)-1A was significantly up-regulated in the P0 Mapt^-/- , but not the Mapt^+/- , pups. These results suggest that tau haploinsufficiency, without the compensation effect of MAP1A, induces reduction of Otx2 expression, increases prenatal cell death, and accordingly leads to selective loss of VTA DA neurons in the early postnatal stage. Our findings highlight the impact of tau haploinsufficiency on the survival of mDANs and indicate that tau may participate in midbrain development in a dose-dependent way.-Zheng, M., Jiao, L., Tang, X., Xiang, X., Wan, X., Yan, Y., Li, X., Zhang, G., Li, Y., Jiang, B., Cai, H., Lin, X. Tau haploinsufficiency causes prenatal loss of dopaminergic neurons in the ventral tegmental area and reduction of transcription factor orthodenticle homeobox 2 expression.

Keywords: MAP1A; compensation effect; midbrain development; tau deficiency.

Figure 1.

Normal mDAN morphology and number in tau-deficient mouse embryos at E14.5. A) TH immunohistochemistry of midbrains of E14.5 Mapt^+/+, Mapt^+/−, and Mapt^−/− mice. Representative coronal sections of midbrain, rostral to caudal, are shown, with white dotted lines demarcating the presumptive (Pre) boundary between the SN and VTA. The lateral TH⁺ area is considered to be Pre-SN, whereas the intermediate is Pre-VTA. Scale bar, 100 μm. B) The number of TH⁺ neurons in the Pre-SN, Pre-VTA, and total midbrain of E14.5 mice (n = 5 for each genotype vs. control littermates). Data are means ± sem.

Figure 2.

The expression levels of the key factors implicated in the mDAN development do not change in tau-deficient mouse embryo brains at E14.5. A) Western blot analysis was used to detect the expression levels of tau, TH, Nurr1, Pitx3, Lmx1b, Shh, Wnt1, and Otx2 in the whole brains of Mapt^+/+, Mapt^+/−, and Mapt^−/− mice at E14.5. B) The bands were quantified by densitometry and normalized to β-actin (n = 6 for each genotype vs. control littermates). Data are means ± sem.

Figure 3.

Heterozygous tau knockout decreases the number of DA neurons in VTA at P0. A) TH immunohistochemistry of midbrains of P0 Mapt^+/+, Mapt^+/−, and Mapt^−/− mice. Representative coronal sections of midbrain from 4.83–5.07 mm rostral (12) are shown, with white dotted lines demarcating the boundary among the VTA, SN, and RRF. Scale bar, 200 μm. B, C) Number of TH⁺ neurons in total midbrains (B) and SN, VTA, and RRF (C) of P0 mice (n = 5 for each genotype vs. control littermates). Data are means ± sem. *P < 0.05.

Figure 4.

TH and Nissl double-immunofluorescence staining in VTA of P0 mice. A) Representative images show TH (green) and Nissl (red) double-immunofluorescence staining in VTA of Mapt^+/+, Mapt^+/−, and Mapt^−/− mice at P0. Scale bar, 100 μm. B) Number of Nissl⁺ neurons in VTA of P0 mice. C) Number of TH and Nissl double-positive neurons in VTA of P0 mice. D) Percentage of TH and Nissl double-positive neurons to the total number of Nissl⁺ neurons in VTA of P0 mice (n = 5 for each genotype vs. control littermates). Data are means ± sem. *P < 0.05.

Figure 5.

Heterozygous tau knockout reduces the expression levels of Otx2 in P0 mouse midbrains. A) Western blot analysis was used to detect the expression levels of tau, TH, Nurr1, Pitx3, Lmx1b, Shh, Wnt1, and Otx2 in the midbrains of Mapt^+/+, Mapt^+/−, and Mapt^−/− mice at P0. B) The bands were quantified by densitometry and normalized to β-actin (n = 6 for each genotype vs. control littermates). Data are means ± sem. *P < 0.05.

Figure 6.

Otx2 expression is reduced in the VTA of Mapt^+/− mice at P0. Aa–f) Representative images show Otx2 (green) immunofluorescence staining in VTA of Mapt^+/+ (Aa, d), Mapt^+/− (Ab, e), and Mapt^−/− (Ac, f) mice at P0. Ad–f) Enlarged areas demarcated by the insets in Aa–c. Ba–f) Representative images show TH (red) immunofluorescence staining in VTA of Mapt^+/+ (Ba, d), Mapt^+/− (Bb, e), and Mapt^−/− (Bc, f) mice at P0. Bd–f) Enlarged areas demarcated by the insets in Ba–c. Ca–f) Representative images show the overlay of Otx2 (Aa–f) and TH (Ba–f) immunofluorescence staining in VTA of Mapt^+/+, Mapt^+/−, and Mapt^−/− mice at P0. Cd–f) Enlarged areas demarcated by the insets in Ca–c). Scale bars, 50 μm (Aa–c, Ba–c, Ca–c); 10 μm (Ad–f, Bd–f, Cd–f). D) Number of Otx2⁺ neurons in VTA of P0 mice. E) Number of Otx2 and TH double-positive neurons in VTA of P0 mice. F) Number of Otx2⁺ neurons in midbrains of E14.5 mice (n = 5 for each genotype vs. control littermates). Data are means ± sem. **P < 0.01.

Figure 7.

Homozygous tau knockout could be compensated by up-regulating expression levels of MAP1A in P0 mouse midbrains. A) Western blot analysis was used to detect the expression levels of MAP1A in the midbrains of Mapt^+/+, Mapt^+/−, and Mapt^−/− mice at P0. B) The bands were quantified by densitometry and normalized to β-tubulin (n = 6 for each genotype vs. control littermates). Data are means ± sem. **P < 0.01.

Figure 8.

Cell death in midbrains of Mapt^+/+, Mapt^+/−, and Mapt^−/− mice at P0, E18.5, and E15.5. A) Representative images show TUNEL (green) and TH (red) double staining in the VTA of E18.5 and P0 Mapt^+/+, Mapt^+/−, and Mapt^−/− mice. Scale bar, 100 μm. The boundary of the VTA is demarcated by white dotted lines. The TUNEL⁺ cells in the VTA are indicated by white dotted circles in each sample. Representative high-magnification merged images of TUNEL and TH double staining are shown in the insets. B) The number of TUNEL⁺ cells in the SN, VTA, and RRF of E18.5 mice. C) The number of TUNEL⁺ cells in the SN, VTA, and RRF of P0 mice. D) The number of TUNEL⁺ cells in Pre-VTA of E15.5 and VTA of E18.5 and P0 mice (n = 5 for each genotype vs. control littermates). Data are means ± sem. *P < 0.05.