Transcription factors Foxa1 and Foxa2 are required for adult dopamine neurons maintenance

Abstract

The proteins Foxa1 and Foxa2 belong to the forkhead family of transcription factors and are involved in the development of several tissues, including liver, pancreas, lung, prostate, and the neural system. Both Foxa1 and Foxa2 are also crucial for the specification and differentiation of dopamine (DA) neurons during embryonic development, while about 30% of mice with an embryonic deletion of a single allele of the Foxa2 gene exhibit an age-related asymmetric loss of DA neurons and develop locomotor symptoms resembling Parkinson's disease (PD). Notably, both Foxa1 and Foxa2 factors continue to be expressed in the adult dopamine system. To directly assess their functions selectively in adult DA neurons, we induced genetic deletions of Foxa1/2 transcription factors in mice using a tamoxifen inducible tissue-specific CreERT2 recombinase expressed under control of the dopamine transporter (DAT) promoter (DATCreERT2). The conditional DA neurons-specific ablation of both genes, but not of Foxa2 alone, in early adulthood, caused a decline of striatal dopamine and its metabolites, along with locomotor deficits. At early pre-symptomatic stages, we observed a decline in aldehyde dehydrogenase family 1, subfamily A1 (Aldh1a1) protein expression in DA neurons. Further analyses revealed a decline of aromatic amino acid decarboxylase (AADC) and a complete loss of DAT expression in these neurons. These molecular changes ultimately led to a reduction of DA neuron numbers in the substantia nigra pars compacta (SNpc) of aged cFoxa1/2 (-/-) mice, resembling the progressive course of PD in humans. Altogether, in this study, we address the molecular, cellular, and functional role of both Foxa1 and Foxa2 factors in the maintenance of the adult dopamine system which may help to find better approaches for PD treatment.

Keywords: Foxa1; Foxa2; Parkinson's disease; dopamine; dopaminergic neurons; neurodegeneration; substantia nigra; transgenic mice.

Figure 1

Absence of changes in locomotor activity and striatal dopamine content upon conditional ablation of Foxa2 gene in adult dopamine neurons. (A) Striatal dopamine content in cFoxa2^−/− mice at indicated time points after TAM treatment (post-TAM). n = 4, 3, 3, 7, 14, 10, 6, 6, 5, 3 for the groups from left to the right on the graph. (B) Latency to fall in the accelerating rotarod assay in control, cFoxa2^+/−, and cFoxa2^−/− mice 6 weeks post-TAM (n = 4, 3, 3, respectively). (C) Latency to fall in the constant speed (35 rpm) rotarod assay in aged control, cFoxa2^+/−, and cFoxa2^−/− mice 58 weeks post-TAM (n = 3, 15, 9, respectively). (D) Reciprocal change of Foxa1 and Foxa2 mRNA levels in the ventral midbrain of control (n = 10) and cFoxa2^−/− (n = 5) mice 24 weeks post-TAM. ^*p < 0.05 in comparison to control, as determined by Student's unpaired t-test.

Figure 2

Locomotor impairments in cFoxa1/2^−/− double mutant mice. (A,B) Latency to fall in the constant speed rotarod assay at 35 rpm (A) and 25 rpm (B) in control and cFoxa1/2^−/− mice (n = 13 and 5, respectively) at indicated time points post-TAM (C–E) The quantification of mean velocity (C) and mean angular velocity (D), and representative running tracks (E) of control, cFoxa2^−/−, cFoxa1^+/−/2^−/−, and cFoxa1/2^−/− mice (n = 11, 6, 10, 5, respectively) in the open field assay performed 21 weeks post-TAM. Length of the open field box side, 60 cm. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 in comparison to control, as determined by Student's unpaired t-test or Two-Way ANOVA followed by Bonferroni post-hoc test. Blue and red dots represent, respectively, the initial and final position of a mouse in the open field assay.

Figure 3

Loss of the Aldh1a1 expression in adult dopamine neurons precedes the onset of locomotor impairments in cFoxa1/2^−/− mice. (A,B) Quantification of tyrosine hydroxylase (TH)-positive neurons in the SNpc (A) or in the VTA (B) of control, cFoxa1^+/−/2^−/−, and cFoxa1/2^−/− mice 24 weeks post-TAM (n = 4, 3, and 5, respectively). (C–F) Quantification of aldehyde dehydrogenase 1 family, member A1 (Aldh1a1)-positive neurons in the ventral midbrain (C,F), SNpc (D) or VTA (E) of control, cFoxa1^+/−/2^−/−, and cFoxa1/2^−/− mice 11 weeks (C) and 24 weeks (D–F) post-TAM expressed relative to the number of TH-positive neurons (n = 4, 3, and 5, respectively). (G) Representative microphotographs of TH (red) and Aldh1a1 (green) immunofluorescent staining and co-localization of these proteins and DAPI (blue) in the ventral midbrain sections from control and cFoxa1/2^−/− mice 24 weeks post-TAM. Scale bar, 200 μm for overviews and 50 μm for insets. ^*p < 0.05, ^**p < 0.01 in comparison to control, as determined by Student's unpaired t-test.

Figure 4

Down-regulation of dopamine neuronal markers in cFoxa1/2^−/− mice. (A–C) Representative microphotographs of tyrosine hydroxylase (TH) (A), aromatic amino acid decarboxylase (AADC) (B) and dopamine transporter (DAT) (C) immunostaining in the ventral midbrain of control and cFoxa1/2^−/− mice 24 weeks post-TAM. Scale bar, 500 μm.

Figure 5

Loss of TH-positive adult dopamine neurons in aged cFoxa1/2^−/− mice. (A) Striatal dopamine content in control or cFoxa1/2^−/− mice 78 weeks after TAM treatment (n = 6 and 3, respectively). (B) Quantification of tyrosine hydroxylase (TH)-positive neurons in the SNpc of control and cFoxa1/2^−/− mice at the same time point (n = 3). (C,D) Representative microphotographs of TH (C) and aromatic amino acid decarboxylase (AADC) (D) immunostaining in the ventral midbrain of control and cFoxa1/2^−/− mice. Scale bar, 500 μm. ^*p < 0.05, ^***p < 0.001 in comparison to control, as determined by Student's unpaired t-test.