The essential role of transcription factor Pitx3 in preventing mesodiencephalic dopaminergic neurodegeneration and maintaining neuronal subtype identities during aging

Pituitary homeobox 3 (Pitx3) is required for the terminal differentiation of nigrostriatal dopaminergic neurons during neuronal development. However, whether Pitx3 contributes to the normal physiological function and cell-type identity of adult neurons remains unknown. To explore the role of Pitx3 in maintaining mature neurons, we selectively deleted Pitx3 in the mesodiencephalic dopaminergic (mdDA) neurons of Pitx3^fl/fl/DAT^CreERT2 bigenic mice using a tamoxifen inducible Cre^ERT2/loxp gene-targeting system. Pitx3^fl/fl/DAT^CreERT2 mice developed age-dependent progressive motor deficits, concomitant with a rapid reduction of striatal dopamine (DA) content and a profound loss of mdDA neurons in the substantia nigra pars compacta (SNc) but not in the adjacent ventral tegmental area (VTA), recapitulating the canonical neuropathological features of Parkinson’s disease (PD). Mechanistic studies showed that Pitx3-deficiency significantly increased the number of cleaved caspase-3⁺ cells in SNc, which likely underwent neurodegeneration. Meanwhile, the vulnerability of SNc mdDA neurons was increased in Pitx3^fl/fl/DAT^CreERT2 mice, as indicated by an early decline in glial cell line-derived neurotrophic factor (GDNF) and aldehyde dehydrogenase 1a1 (Aldh1a1) levels. Noticeably, somatic accumulation of α-synuclein (α-syn) was also significantly increased in the Pitx3-deficient neurons. Together, our data demonstrate that the loss of Pitx3 in fully differentiated mdDA neurons results in progressive neurodegeneration, indicating the importance of the Pitx3 gene in adult neuronal survival. Our findings also suggest that distinct Pitx3-dependent pathways exist in SNc and VTA mdDA neurons, correlating with the differential vulnerability of SNc and VTA mdDA neurons in the absence of Pitx3.Subject terms: Neuroscience, Neurological disorders     

SLC35D3 increases autophagic activity in midbrain dopaminergic neurons by enhancing BECN1-ATG14-PIK3C3 complex formation

Searching for new regulators of autophagy involved in selective dopaminergic (DA) neuron loss is a hallmark in the pathogenesis of Parkinson disease (PD). We here report that an endoplasmic reticulum (ER)-associated transmembrane protein SLC35D3 is selectively expressed in subsets of midbrain DA neurons in about 10% TH (tyrosine hydroxylase)-positive neurons in the substantia nigra pars compacta (SNc) and in about 22% TH-positive neurons in the ventral tegmental area (VTA). Loss of SLC35D3 in ros (roswell mutant) mice showed a reduction of 11.9% DA neurons in the SNc and 15.5% DA neuron loss in the VTA with impaired autophagy. We determined that SLC35D3 enhanced the formation of the BECN1-ATG14-PIK3C3 complex to induce autophagy. These results suggest that SLC35D3 is a new regulator of tissue-specific autophagy and plays an important role in the increased autophagic activity required for the survival of subsets of DA neurons.     

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.     

Heterogeneous Distribution of Kir3 Potassium Channel Proteins Within Dopaminergic Neurons in the Mesencephalon of the Rat Brain

1. Dopaminergic neurons in the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA) of the ventral mesencephalon play an important role in the regulation of the parallel basal ganglia loops. 2. We have raised affinity-purified polyclonal rabbit antibodies specific for all four members of the Kir3 family of inwardly rectifying potassium channels (Kir3.1–Kir3.4) to investigate the distribution of the channel proteins in the dopaminergic neurons of the rat mesencephalon at light and electron microscopic level. In addition, immunocytochemical double labeling with tyrosine hydroxylase (TH), a marker of dopaminergic neurons, were performed. 3. All Kir3 channels were present in this region. However, the individual proteins showed differential cellular and subcellular distributions. 4. Kir3.1 immunoreactivity was found in SNc fibers and some neurons of the substantia nigra pars reticulata (SNr). Few Kir3.3-positive neurons were found in the SNc. However, a strong Kir3.3 signal was identified in the SNr neuropil. Weak Kir3.4 staining was detected in neuronal somata as well as in dendritic fibers of both parts of the SN. 5. In the VTA, Kir3.1, Kir3.3, and Kir3.4 showed only weak staining of neuropil structures. The distribution of the Kir3.2 channel protein was especially striking with strong labeling in the SNc and in the lateral but not central VTA. 6. Our results suggest that the heterogeneously distributed Kir3.2 channel proteins could help to discriminate the dopaminergic neurons of VTA and SNc.     

ApoER2 and VLDLr Are Required for Mediating Reelin Signalling Pathway for Normal Migration and Positioning of Mesencephalic Dopaminergic Neurons

The migration of mesencephalic dopaminergic (mDA) neurons from the subventricular zone to their final positions in the substantia nigra compacta (SNc), ventral tegmental area (VTA), and retrorubral field (RRF) is controlled by signalling from neurotrophic factors, cell adhesion molecules (CAMs) and extracellular matrix molecules (ECM). Reelin and the cytoplasmic adaptor protein Disabled-1 (Dab1) have been shown to play important roles in the migration and positioning of mDA neurons. Mice lacking Reelin and Dab1 both display phenotypes characterised by the failure of nigral mDA neurons to migrate properly. ApoER2 and VLDLr are receptors for Reelin signalling and are therefore part of the same signal transduction pathway as Dab1. Here we describe the roles of ApoER2 and VLDLr in the proper migration and positioning of mDA neurons in mice. Our results demonstrate that VLDLr- and ApoER2-mutant mice have both a reduction in and abnormal positioning of mDA neurons. This phenotype was more pronounced in VLDLr-mutant mice. Moreover, we provide evidence that ApoER2/VLDLr double-knockout mice show a phenotype comparable with the phenotypes observed for Reelin- and Dab1- mutant mice. Taken together, our results demonstrate that the Reelin receptors ApoER2 and VLDLr play essential roles in Reelin-mediated migration and positioning of mDA neurons.     

Mesencephalic dopaminergic neurons express a repertoire of olfactory receptors and respond to odorant-like molecules

Background

The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the Substantia Nigra (SN) (A9 neurons) and the Ventral Tegmental Area (VTA) (A10 cells). Selective degeneration of A9 neurons occurs in Parkinson’s disease (PD) while abnormal function of A10 cells has been linked to schizophrenia, attention deficit and addiction. The molecular basis that underlies selective vulnerability of A9 and A10 neurons is presently unknown.

Results

By taking advantage of transgenic labeling, laser capture microdissection coupled to nano Cap-Analysis of Gene Expression (nanoCAGE) technology on isolated A9 and A10 cells, we found that a subset of Olfactory Receptors (OR)s is expressed in mDA neurons. Gene expression analysis was integrated with the FANTOM5 Helicos CAGE sequencing datasets, showing the presence of these ORs in selected tissues and brain areas outside of the olfactory epithelium. OR expression in the mesencephalon was validated by RT-PCR and in situ hybridization. By screening 16 potential ligands on 5 mDA ORs recombinantly expressed in an heterologous in vitro system, we identified carvone enantiomers as agonists at Olfr287 and able to evoke an intracellular Ca²⁺ increase in solitary mDA neurons. ORs were found expressed in human SN and down-regulated in PD post mortem brains.

Conclusions

Our study indicates that mDA neurons express ORs and respond to odor-like molecules providing new opportunities for pharmacological intervention in disease.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-729) contains supplementary material, which is available to authorized users.     

Pitx3 is required for motor activity and for survival of a subset of midbrain dopaminergic neurons

Mesencephalic dopaminergic (MesDA) neurons play crucial roles in motor and behavioral processes; their loss in Parkinson's disease (PD) results in striatal dopamine (DA) deficiency and hypokinetic movement disorder. The Pitx3 homeobox gene is expressed in the MesDA system. We now show that only a subset of MesDA neurons express Pitx3 and that in Pitx3-deficient aphakia mice, this subset is progressively lost by apoptosis during fetal (substantia nigra, SN) and postnatal (ventral tegmental area) development, resulting in very low striatal DA and akinesia. Similar to human PD, dorsal SN neurons (which are Pitx3 negative) are spared in mutant mice. Thus, Pitx3 defines a pathway for survival of neurons that are implicated in PD and that are required for spontaneous locomotor activity.     

Pitx3 regulates tyrosine hydroxylase expression in the substantia nigra and identifies a subgroup of mesencephalic dopaminergic progenitor neurons during mouse development

Recent studies of mouse mutant aphakia have implicated the homeobox gene Pitx3 in the survival of substantia nigra dopaminergic neurons, the degeneration of which causes Parkinson's disease. To directly investigate a role for Pitx3 in midbrain DA neuron development, we have analysed a line of Pitx3-null mice that also carry an eGFP reporter under the control of the endogenous Pitx3 promoter. We show that the lack of Pitx3 resulted in a loss of nascent substantia nigra dopaminergic neurons at the beginning of their final differentiation. Pitx3 deficiency also caused a loss of tyrosine hydroxylase (TH) expression specifically in the substantia nigra neurons. Therefore, our study provides the first direct evidence that the aphakia allele of Pitx3 is a hypomorph and that Pitx3 is required for the regulation of TH expression in midbrain dopaminergic neurons as well as the generation and/or maintenance of these cells. Furthermore, using the targeted GFP reporter as a midbrain dopaminergic lineage marker, we have identified previously unrecognised ontogenetically distinct subpopulations of dopaminergic cells within the ventral midbrain based on their temporal and topographical expression of Pitx3 and TH. Such an expression pattern may provide the molecular basis for the specific dependence of substantia nigra DA neurons on Pitx3.     

Expression of Trefoil Factor 1 in the Developing and Adult Rat Ventral Mesencephalon

Trefoil factor 1 (TFF1) belongs to a family of secreted peptides with a characteristic tree-looped trefoil structure. TFFs are mainly expressed in the gastrointestinal tract where they play a critical role in the function of the mucosal barrier. TFF1 has been suggested as a neuropeptide, but not much is known about its expression and function in the central nervous system. We investigated the expression of TFF1 in the developing and adult rat midbrain. In the adult ventral mesencephalon, TFF1-immunoreactive (-ir) cells were predominantly found in the substantia nigra pars compacta (SNc), the ventral tegmental area (VTA) and in periaqueductal areas. While around 90% of the TFF1-ir cells in the SNc co-expressed tyrosine hydroxylase (TH), only a subpopulation of the TH-ir neurons expressed TFF1. Some TFF1-ir cells in the SNc co-expressed the calcium-binding proteins calbindin or calretinin and nearly all were NeuN-ir confirming a neuronal phenotype, which was supported by lack of co-localization with the astroglial marker glial fibrillary acidic protein (GFAP). Interestingly, at postnatal (P) day 7 and P14, a significantly higher proportion of TH-ir neurons in the SNc co-expressed TFF1 as compared to P21. In contrast, the proportion of TFF1-ir cells expressing TH remained unchanged during postnatal development. Furthermore, significantly more TH-ir neurons expressed TFF1 in the SNc, compared to the VTA at all four time-points investigated. Injection of the tracer fluorogold into the striatum of adult rats resulted in retrograde labeling of several TFF1 expressing cells in the SNc showing that a significant fraction of the TFF1-ir cells were projection neurons. This was also reflected by unilateral loss of TFF1-ir cells in SNc of 6-hydroxylase-lesioned hemiparkinsonian rats. In conclusion, we show for the first time that distinct subpopulations of midbrain dopaminergic neurons express TFF1, and that this expression pattern is altered in a rat model of Parkinson’s disease.