Simone M. Smits

Early developmental failure of substantia nigra dopamine neurons in mice lacking the homeodomain gene Pitx3

The mesencephalic dopamine (mesDA) system is involved in the control of movement and behavior. The expression of Pitx3 in the brain is restricted to the mesDA system and the gene is induced relatively late, at E11.5, a time when tyrosine hydroxylase (Th) gene expression is initiated. We show here that, in the Pitx3-deficient aphakia (ak) mouse mutant, the mesDA system is malformed. Owing to the developmental failure of mesDA neurons in the lateral field of the midbrain, mesDA neurons are not found in the SNc and the projections to the caudate putamen are selectively lost. However, Pitx3 is expressed in all mesDA neurons in control animals. Therefore, mesDA neurons react specifically to the loss of Pitx3. Defects of motor control where not seen in the ak mice, suggesting that other neuronal systems compensate for the absence of the nigrostriatal pathway. However, an overall lower activity was observed. The results suggest that Pitx3 is specifically required for the formation of the SNc subfield at the onset of dopaminergic neuron differentiation.

Involvement of Nurr1 in specifying the neurotransmitter identity of ventral midbrain dopaminergic neurons

The mesencephalic dopaminergic (mesDA) system is involved in many brain functions including motor control and motivated behaviour, and is of clinical importance because of its implication in psychiatric disorders and Parkinsons disease. Nurr1, a member of the nuclear hormone receptor superfamily of transcription factors, is essential for establishing the dopaminergic phenotype, because expression of tyrosine hydroxylase (TH), the rate‐limiting enzyme in dopamine synthesis, requires Nurr1. In addition, Nurr1 plays an important role in the maintenance of mesDA neurons. Neonatal Nurr1 knockout mice lack expression of the dopamine transporter (DAT), the vesicular monoamine transporter 2 (VMAT2) and l‐aromatic amino acid decarboxylase (AADC) in addition to TH specifically in mesDA neurons. It is unclear whether the lack of expression of these dopaminergic markers is caused by a maintenance defect or whether the induction of these markers depends on Nurr1 expression. To address this problem, the expression of DAT, VMAT2 and AADC was analysed at embryonic day 12.5 and 14.5. Here we demonstrate that induction of VMAT2 and DAT specifically in mesDA neurons requires Nurr1 expression, whereas AADC expression in mesDA neurons is induced independently of Nurr1 function.

Retinoic acid counteracts developmental defects in the substantia nigra caused by Pitx3 deficiency.

Selective neuronal loss in the substantia nigra (SNc), as described for Parkinsons disease (PD) in humans and for Pitx3 deficiency in mice, highlights the existence of neuronal subpopulations. As yet unknown subset-specific gene cascades might underlie the observed differences in neuronal vulnerability. We identified a developmental cascade in mice in which Ahd2 (Aldh1a1) is under the transcriptional control of Pitx3. Interestingly, Ahd2 distribution is restricted to a subpopulation of the meso-diencephalic dopaminergic (mdDA) neurons that is affected by Pitx3 deficiency. Ahd2 is involved in the synthesis of retinoic acid (RA), which has a crucial role in neuronal patterning, differentiation and survival in the brain. Most intriguingly, restoring RA signaling in the embryonic mdDA area counteracts the developmental defects caused by Pitx3 deficiency. The number of tyrosine hydroxylase-positive (TH+) neurons was significantly increased after RA treatment in the rostral mdDA region of Pitx3-/- embryos. This effect was specific for the rostral part of the developing mdDA area, and was observed exclusively in Pitx3-/- embryos. The effect of RA treatment during the critical phase was preserved until later in development, and our data suggest that RA is required for the establishment of proper mdDA neuronal identity. This positions Pitx3 centrally in a mdDA developmental cascade linked to RA signaling. Here, we propose a novel mechanism in which RA is involved in mdDA neuronal development and maintenance, providing new insights into subset-specific vulnerability in PD.

Homeobox gene Pitx3 and its role in the development of dopamine neurons of the substantia nigra.

The homeobox gene Pitx3 plays an important part in the development and function of vertebrate midbrain dopaminergic neurons. Re-localization of the genetic defect in the mouse mutant aphakia to the Pitx3 locus, together with the subsequent identification of two deletions causing the gene to be silent, has been the hallmark of several studies into the role of Pitx3. In this review, we summarize the data and reflect on the role of Pitx3 in the development of dopamine neurons in the midbrain. The data indicate that Pitx3 is essential for the survival of dopamine neurons located in the substantia nigra compacta during development. Molecular analysis of the underlying mechanisms might provide new insights for understanding the selective degeneration observed in Parkinson patients.

Transcription Factors in the Development of Midbrain Dopamine Neurons

Abstract: The development of midbrain dopamine (DA) neurons follows a number of stages marked by distinct events. After preparation of the region by signals that provide induction and patterning, at least two cascades of transcription factors contribute to the fully matured midbrain DA systems. One cascade involving the nuclear receptor Nurr1 is required to synthesize the neurotransmitter DA; the enzyme tyrosine hydroxylase (TH) depends on it. The other cascade involves homeobox genes. Lmx1b and engrailed genes are expressed before the genesis of DA neurons and maintain their expression in these neurons. Lmx1b drives Ptx3, which is the latest transcription factor known to be induced. Its induction coincides with that of TH. Disruption of the function of Ptx3 affects the formation of the substantia nigra (SN) and alters the anatomical organization of the midbrain DA systems. While each cascade contributes to a specific aspect of DA neurons, both cascades are required for survival during development, indicating that the maintenance of DA neurons is delicately dependent on the appropriate activity of multiple transcriptional cascades.

The role of Pitx3 in survival of midbrain dopaminergic neurons

Dopamine belongs to the most intensively studied neurotransmitters of the brain, because of its implications in psychiatric and neurological disorders. Although, clinical relevance of midbrain dopaminergic (mDA) neurons is well recognized and dopaminergic dysfunction may have a genetic component, the genetic cascades underlying developmental processes are still largely unknown. With the advances in molecular biology, mDA neurons and their involvement in psychiatric and neurological disorders are now subject of studies that aim to delineate the fundamental neurobiology of these neurons. These studies are concerned with developmental processes, cell-specific gene expression and regulation, molecular pharmacology, and genetic association of dopamine-related genes and mDA-associated disorders. Several transcription factors implicated in the post-mitotic mDA development, including Nurr1, Lmx1b, Pitx3, and En1/En2 have contributed to the understanding of how mDA neurons are generated in vivo. Furthermore, these studies provide insights into new strategies for future therapies of Parkinsons Disease (PD) using stem cells for engineering DA neurons in vitro. Here, we will discuss the role of Pitx3 in molecular mechanisms involved in the regional specification, neuronal specification and differentiation of mDA neurons.

Molecular and cellular alterations in the Pitx3-deficient midbrain dopaminergic system

Parkinsons disease (PD) is a progressive neurodegenerative disorder characterized by loss of midbrain dopaminergic (mDA) neurons in the substantia nigra compacta (SNc). In order to provide insights into adaptive mechanisms of the mDA system in pathology, specific molecular and cellular parameters of the mDA system were studied in Pitx3-deficient Aphakia (ak) mice, which suffer from severe developmental failure of SNc mDA neurons. Here, we demonstrate differential changes in striatal gene expression, reflecting the specific neuronal loss in these mice. In addition, the neuronal activity of remaining mDA neurons in the ventral tegmental area (VTA) was significantly increased in ak mice. In conclusion, ak mice display specific molecular and cellular alterations in the mDA system that provide new insights in compensatory mechanisms present in mDA-associated disorders such as PD.

Molecular Marker Differences Relate to Developmental Position and Subsets of Mesodiencephalic Dopaminergic Neurons

The development of mesodiencephalic dopaminergic (mdDA) neurons located in the substantia nigra compacta (SNc) and ventral tegmental area (VTA) follow a number of stages marked by distinct events. After preparation of the region by signals that provide induction and patterning, several transcription factors have been identified, which are involved in specifying the neuronal fate of these cells. The specific vulnerability of SNc neurons is thought to root in these specific developmental programs. The present study examines the positions of young postmitotic mdDA neurons to relate developmental position to mdDA subset specific markers. MdDA neurons were mapped relative to the neuromeric domains (prosomeres 1-3 (P1-3), midbrain, and hindbrain) as well as the longitudinal subdivisions (floor plate, basal plate, alar plate), as proposed by the prosomeric model. We found that postmitotic mdDA neurons are located mainly in the floorplate domain and very few in slightly more lateral domains. Moreover, mdDA neurons are present along a large proportion of the anterior/posterior axis extending from the midbrain to P3 in the diencephalon. The specific positions relate to some extent to the presence of specific subset markers as Ahd2. In the adult stage more of such subsets specific expressed genes are present and may represent a molecular map defining molecularly distinct groups of mdDA neurons.

Alterations in serotonin signalling are involved in the hyperactivity of Pitx3‐deficient mice

Pitx3 deficiency in mice causes a dramatic loss of dopaminergic neurones located in the substantia nigra pars compacta during development. This early disruption of the nigrostriatal pathway in Pitx3‐deficient mice is characterized by increased spontaneous home‐cage activity levels during the habitual sleep phase of these animals. These findings are reminiscent of the spontaneous hyperactivity in mice neonatally lesioned with 6‐hydroxydopamine, which is caused by an extensive serotonergic hyperinnervation of the striatum. The present study investigated whether an imbalance between dopamine (DA) and serotonin (5‐HT) signalling is involved in the behavioural phenotype of Pitx3‐deficient mice. Serotonergic hyperinnervation was demonstrated by increased [3H]‐citalopram autoradiographic binding specifically in the dorsal striatum of adult Pitx3‐deficient mice, indicating alterations in 5‐HT transporter levels that correlated to DA dysfunction in Pitx3 deficiency. In addition, stimulus‐induced release of DA and 5‐HT indicated an altered balance between these neurotransmitters in the dorsal striatum of Pitx3–/– mice. To determine whether the increased 5‐HT signalling was involved in the spontaneous hyperactivity during the light phase observed in Pitx3 deficiency, we treated Pitx3‐deficient and control mice with the selective irreversible tryptophan hydroxylase inhibitor p‐chlorophenylalanine to decrease 5‐HT levels. Reduction of 5‐HT levels in Pitx3‐deficient mice decreased their locomotor activity to normal levels, whereas the same treatment increased the locomotor activity levels of control mice. Taken together, our results indicate alterations in 5‐HT signalling in Pitx3‐deficient mice that underlie their spontaneous hyperactivity.

Strategies to unravel molecular codes essential for the development of meso-diencephalic dopaminergic neurons

Understanding the development of neuronal systems has become an important asset in the attempt to solve complex questions about neuropathology as found in Parkinsons disease, schizophrenia and other complex neuronal diseases. The development of anatomical and functional divergent structures in the brain is achieved by a combination of early anatomical patterning and highly coordinated neuronal migration and differentiation events. Fundamental to the existence of divergent structures in the brain is the early region‐specific molecular programming. Neuronal progenitors located along the neural tube can still adapt many different identities. Their exact position in the developing brain, however, determines early molecular specification by region‐specific signalling molecules. These signals determine time and region‐specific expression of early regulatory genes, leading to neuronal differentiation. Here, we focus on a well‐described neuronal group, the meso‐diencephalic dopaminergic neurons, of which heterogeneity based on anatomical position could account for the difference in vulnerability of specific subgroups as observed in Parkinsons disease. The knowledge of their molecular coding helps us to understand how the meso‐diencephalic dopaminergic system is built and could provide clues that unravel mechanisms associated with the neuropathology in complex diseases such as Parkinsons disease.