DYRK1A promotes dopaminergic neuron survival in the developing brain and in a mouse model of Parkinson's disease

In the brain, programmed cell death (PCD) serves to adjust the numbers of the different types of neurons during development, and its pathological reactivation in the adult leads to neurodegeneration. Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) is a pleiotropic kinase involved in neural proliferation and cell death, and its role during brain growth is evolutionarily conserved. Human DYRK1A lies in the Down syndrome critical region on chromosome 21, and heterozygous mutations in the gene cause microcephaly and neurological dysfunction. The mouse model for DYRK1A haploinsufficiency (the Dyrk1a^+/− mouse) presents neuronal deficits in specific regions of the adult brain, including the substantia nigra (SN), although the mechanisms underlying these pathogenic effects remain unclear. Here we study the effect of DYRK1A copy number variation on dopaminergic cell homeostasis. We show that mesencephalic DA (mDA) neurons are generated in the embryo at normal rates in the Dyrk1a haploinsufficient model and in a model (the mBACtgDyrk1a mouse) that carries three copies of Dyrk1a. We also show that the number of mDA cells diminishes in postnatal Dyrk1a^+/− mice and increases in mBACtgDyrk1a mice due to an abnormal activity of the mitochondrial caspase9 (Casp9)-dependent apoptotic pathway during the main wave of PCD that affects these neurons. In addition, we show that the cell death induced by 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), a toxin that activates Casp9-dependent apoptosis in mDA neurons, is attenuated in adult mBACtgDyrk1a mice, leading to an increased survival of SN DA neurons 21 days after MPTP intoxication. Finally, we present data indicating that Dyrk1a phosphorylation of Casp9 at the Thr125 residue is the mechanism by which this kinase hinders both physiological and pathological PCD in mDA neurons. These data provide new insight into the mechanisms that control cell death in brain DA neurons and they show that deregulation of developmental apoptosis may contribute to the phenotype of patients with imbalanced DYRK1A gene dosage.The total number of neurons in the brain, and ultimately the size of this organ, depends both on the number of cells that are produced during neurogenesis and the number of neurons that die due to physiological programmed cell death (PCD). Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) regulates brain growth in a dose-dependent manner,¹ and indeed, loss-of-function mutations in DYRK1A (minibrain in Drosophila melanogaster) cause microcephaly and several neurological alterations in humans,^{2, 3, 4, 5} mice⁶ and flies.⁷ Accordingly, it has been proposed that haploinsufficiency of DYRK1A is the cause of the microcephaly and developmental delay associated to partial monosomy of chromosome 21 involving DYRK1A.⁸ Moreover, triplication of the gene has been associated to the developmental brain dysfunctions and age-associated neurodegeneration observed in Down syndrome.^{9, 10, 11}Anatomical analysis of adult Dyrk1a mutant mice that model human diseases involving an imbalance in DYRK1A gene dosage (the Dyrk1a^+/− mouse and the mBACtgDyrk1a mouse, carrying one or three functional copies of Dyrk1a, respectively) revealed a positive correlation between Dyrk1a gene copy number, the overall size of the brain and the number of neurons in specific regions.¹ DYRK1A regulates several fundamental neurodevelopmental processes, including proliferation, neuron differentiation and PCD.¹² Overexpression of DYRK1A in neural precursors attenuates proliferation and promotes the differentiation of neurons in different model systems.^{13, 14, 15} Conversely, treatment of neural progenitors with DYRK1A kinase inhibitors increases proliferation.¹⁵ Although these data are consistent with some of the defects in cellularity identified in specific brain regions of Dyrk1a gene copy number mutants, they cannot explain the severe microcephaly evident in mice and humans carrying one functional copy of DYRK1A, or the overall macrocephaly in the mBACtgDyrk1a model carrying three Dyrk1a alleles.^{1, 5} Thus, deregulation of other DYRK1A functions might also contribute to the defects in brain cellularity in these Dyrk1a gene copy number mutants, such as those described in retinal neurons that restrain developmental PCD.¹⁶Dopaminergic (DA) neurons in the substantia nigra (SN) and ventral tegmental area (VTA) have an important role in controlling fine motor actions, as well as in motivation and reward behaviours, and their loss is associated with Parkinson''s disease.¹⁷ In aged Dyrk1a^+/− mice the SN is smaller and contains fewer DA neurons than in wild-type mice.¹⁸ These mutant animals are hypoactive, with altered gait dynamics, and as these defects are evident preweaning and in young animals,^{6, 18, 19} as well as in children with heterozygous mutations in DYRK1A,^{3, 4, 5} they might arise during development.To provide insight into the aetiology of the neurological phenotype caused by DYRK1A haploinsufficiency, here we studied the development of mesencephalic DA (mDA) neurons in Dyrk1a^+/− and mBACtgDyrk1a mouse models. The results obtained show that Dyrk1a copy number variation does not affect the generation of DA neurons, but rather it modifies the number of these neurons that undergo physiological PCD due to an inhibitory effect of the Dyrk1a kinase on the apoptotic activity of caspase9 (Casp9), the initiator caspase in the mitochondrial-dependent apoptotic pathway.²⁰ Thus, deregulation of Casp9-dependent PCD during development may contribute to the brain size defects observed in aneuploidies involving DYRK1A.As inappropriate re-activation of the mitochondrial-dependent apoptotic pathway in mature mDA neurons contributes to the neurodegeneration associated with Parkinson''s disease,²¹ we used the mBACtgDyrk1a mouse model to assess whether basal Dyrk1a-dependent inhibition of Casp9 apoptotic activity could restrain the neurodegeneration induced in vivo by the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Our results show that the apoptotic response to the toxin in mBACtgDyrk1a mice is significantly attenuated, leading to an increase in the number of SN pars compacta DA neurons that resist the pathological insult.