The atypical homeoprotein Pbx1a participates in the axonal pathfinding of mesencephalic dopaminergic neurons.

BACKGROUND: The pre B-cell leukemia transcription factor 1 (Pbx1) genes belong to the three amino acid loop extension family of homeodomain proteins that form hetero-oligomeric complexes with other homeodomain transcription factors, thereby modulating target specificity, DNA binding affinity and transcriptional activity of their molecular associates. RESULTS: Here, we provide evidence that Pbx1 is expressed in mesencephalic dopaminergic neurons from embryonic day 11 into adulthood and determines some of the cellular properties of this neuronal population. In Pbx1-deficient mice, the mesencephalic dopaminergic axons stall during mid-gestation at the border between di- and telencephalon before entering the ganglionic eminence, leading to a loose organization of the axonal bundle and partial misrouting. In Pbx1-deficient dopaminergic neurons, the high affinity netrin-1 receptor, deleted in colon cancer (DCC), is down-regulated. Interestingly, we found several conserved Pbx1 binding sites in the first intron of DCC, suggesting a direct regulation of DCC transcription by Pbx1. CONCLUSIONS: The expression of Pbx1 in dopaminergic neurons and its regulation of DCC expression make it an important player in defining the axonal guidance of the midbrain dopaminergic neurons, with possible implications for the normal physiology of the nigro-striatal system as well as processes related to the degeneration of neurons during the course of Parkinson's disease.

Parkinson's disease candidate gene prioritization based on expression profile of midbrain dopaminergic neurons.

BACKGROUND: Parkinson's disease is the second most common neurodegenerative disorder. The pathological hallmark of the disease is degeneration of midbrain dopaminergic neurons. Genetic association studies have linked 13 human chromosomal loci to Parkinson's disease. Identification of gene(s), as part of the etiology of Parkinson's disease, within the large number of genes residing in these loci can be achieved through several approaches, including screening methods, and considering appropriate criteria. Since several of the identified Parkinson's disease genes are expressed in substantia nigra pars compact of the midbrain, expression within the neurons of this area could be a suitable criterion to limit the number of candidates and identify PD genes. METHODS: In this work we have used the combination of findings from six rodent transcriptome analysis studies on the gene expression profile of midbrain dopaminergic neurons and the PARK loci in OMIM (Online Mendelian Inheritance in Man) database, to identify new candidate genes for Parkinson's disease. RESULTS: Merging the two datasets, we identified 20 genes within PARK loci, 7 of which are located in an orphan Parkinson's disease locus and one, which had been identified as a disease gene. In addition to identifying a set of candidates for further genetic association studies, these results show that the criteria of expression in midbrain dopaminergic neurons may be used to narrow down the number of genes in PARK loci for such studies.

Elevated P75NTR expression causes death of engrailed-deficient midbrain dopaminergic neurons by Erk1/2 suppression.

BACKGROUND: The homeodomain transcription factors Engrailed-1 and Engrailed-2 are required for the survival of mesencephalic dopaminergic (mesDA) neurons in a cell-autonomous and gene-dose-dependent manner. Homozygote mutant mice, deficient of both genes (En1-/-;En2-/-), die at birth and exhibit a loss of all mesDA neurons by mid-gestation. In heterozygote animals (En1+/-;En2-/-), which are viable and fertile, postnatal maintenance of the nigrostriatal dopaminergic system is afflicted, leading to a progressive degeneration specific to this subpopulation and Parkinson's disease-like molecular and behavioral deficits. RESULTS: In this work, we show that the dose of Engrailed is inversely correlated to the expression level of the pan-neurotrophin receptor gene P75NTR (Ngfr). Loss of mesDA neurons in the Engrailed-null mutant embryos is caused by elevated expression of this neurotrophin receptor: Unusually, in this case, the cell death signal of P75NTR is mediated by suppression of Erk1/2 (extracellular-signal-regulated kinase 1/2) activity. The reduction in expression of Engrailed, possibly related to the higher levels of P75NTR, also decreases mitochondrial stability. In particular, the dose of Engrailed determines the sensitivity to cell death induced by the classic Parkinson-model toxin MPTP and to inhibition of the anti-apoptotic members of the Bcl-2 family of proteins. CONCLUSION: Our study links the survival function of the Engrailed genes in developing mesDA neurons to the regulation of P75NTR and the sensitivity of these neurons to mitochondrial insult. The similarities to the disease etiology in combination with the nigral phenotype of En1+/-;En2-/- mice suggests that haplotype variations in the Engrailed genes and/or P75NTR that alter their expression levels could, in part, determine susceptibility to Parkinson's disease.

Linkage of cDNA expression profiles of mesencephalic dopaminergic neurons to a genome-wide in situ hybridization database.

Midbrain dopaminergic neurons are involved in control of emotion, motivation and motor behavior. The loss of one of the subpopulations, substantia nigra pars compacta, is the pathological hallmark of one of the most prominent neurological disorders, Parkinson's disease. Several groups have looked at the molecular identity of midbrain dopaminergic neurons and have suggested the gene expression profile of these neurons. Here, after determining the efficiency of each screen, we provide a linked database of the genes, expressed in this neuronal population, by combining and comparing the results of six previous studies and verification of expression of each gene in dopaminergic neurons, using the collection of in situ hybridization in the Allen Brain Atlas.

Transcriptional regulation of mesencephalic dopaminergic neurons: the full circle of life and death.

Since mesencephalic dopaminergic neurons are associated to one of the most prominent human neurodegenerative ailments, Parkinson's disease, the molecular mechanism underlying their development and adult cellular properties has been the subject of intense investigations. Throughout life, transcription factors determine the fate of this neuronal population and control essential processes such as localization in the ventral midbrain, their neurotransmitter phenotype, their target innervations and synapse formation. Studies of transcription factors, such as Nurr1, Pitx3, Engrailed-1/2, and Lmx1a/b, have not only revealed importance of these genes during development, but also roles in the long-term survival and maintenance of these neurons. In this review, we will discuss the function of these transcription factors throughout the life of mesencephalic dopaminergic neurons and their value in the study of the disease mechanism.

Merging mouse transcriptome analyses with Parkinson's disease linkage studies.

The hallmark of Parkinson's disease (PD OMIM #168600) is the degeneration of the nigral dopaminergic system affecting approximately 1% of the human population older than 65. In pursuit of genetic factors contributing to PD, linkage and association studies identified several susceptibility genes. The majority of these genes are expressed by the dopamine-producing neurons in the substantia nigra. We, therefore, propose expression by these neurons as a selection criterion, to narrow down, in a rational manner, the number of candidate genes in orphan PD loci, where no mutation has been associated thus far. We determined the corresponding human chromosome locations of 1435 murine cDNA fragments obtained from murine expression analyses of nigral dopaminergic neurons and combined these data with human linkage studies. These fragments represent 19 genes within orphan OMIM PD loci. We used the same approach for independent association studies and determined the genes in neighborhood to the peaks with the highest LOD score value. Our approach did not make any assumptions about disease mechanisms, but it, nevertheless, revealed alpha-synuclein, NR4A2 (Nurr1), and the tau genes, which had previously been associated to PD. Furthermore, our transcriptome analysis identified several classes of candidate genes for PD mutations and may also provide insight into the molecular pathways active in nigral dopaminergic neurons.

Slow progressive degeneration of nigral dopaminergic neurons in postnatal Engrailed mutant mice.

The homeobox transcription factors Engrailed-1 and Engrailed-2 are required for the survival of mesencephalic dopaminergic neurons in a cell-autonomous and gene-dose-dependent manner. Because of this requirement, the cells die by apoptosis when all four alleles of the Engrailed genes are genetically ablated (En1-/-;En2-/-). In the present study, we show that viable and fertile mice, heterozygous null for Engrailed-1 and homozygous null for Engrailed-2 (En1+/-;En2-/-), have an adult phenotype that resembles key pathological features of Parkinson's disease. Specifically, postnatal mutant mice exhibit a progressive degeneration of dopaminergic neurons in the substantia nigra during the first 3 mo of their lives, leading to diminished storage and release of dopamine in the caudate putamen, motor deficits similar to akinesia and bradykinesia, and a lower body weight. This genetic model may provide access to the molecular etiology for Parkinson's disease and could assist in the development of novel treatments for this neurodegenerative disorder.

One step cloning of defined DNA fragments from large genomic clones.

Recently, the nucleotide sequences of entire genomes became available. This information combined with older sequencing data discloses the exact chromosomal location of millions of nucleotide markers stored in the databases at NCBI, EMBO or DDBJ. Despite having resolved the intron/exon structures of all described genes within these genomes with a stroke of a pen, the sequencing data opens up other interesting possibilities. For example, the genomic mapping of the end sequences of the human, murine and rat BAC libraries generated at The Institute for Genomic Research (TIGR), reveals now the entire encompassed sequence of the inserts for more than a million of these clones. Since these clones are individually stored, they are now an invaluable source for experiments which depend on genomic DNA. Isolation of smaller fragments from such clones with standard methods is a time consuming process. We describe here a reliable one-step cloning technique to obtain a DNA fragment with a defined size and sequence from larger genomic clones in less than 48 hours using a standard vector with a multiple cloning site, and common restriction enzymes and equipment. The only prerequisites are the sequences of ends of the insert and of the underlying genome.

Engrailed genes control developmental fate of serotonergic and noradrenergic neurons in mid- and hindbrain in a gene dose-dependent manner.

In vertebrates and insects, the homeobox transcription factors of the engrailed family have a dual function. They take part in regionalization during early embryogenesis and later in neuronal specification. In mammals, two engrailed homologues exist, engrailed-1 and engrailed-2, which are expressed in a broad band around the isthmus at an age when the serotonergic and noradrenergic neurons in mid/hindbrain are generated. The analysis of engrailed-1 and -2 double mutant mice revealed a specific, redundant, and gene dose-dependent requirement of the two transcription factors for the development of the serotonergic dorsal raphe nucleus and the noradrenergic locus caeruleus. Both nuclei are lost in engrailed double mutant mice; however, directly adjacent nuclei of the same neurotransmitter phenotype are not affected. An almost identical phenotype is found in mutant mice null for Wnt1, indicating that the engrailed genes provide essential positional information for the development of the two nuclei during early embryogenesis.

The neuregulin receptor, ErbB4, is not required for normal development and adult maintenance of the substantia nigra pars compacta.

Degeneration of dopaminergic neurons in the substantia nigra is associated with one of the most prominent human neurological disorders, Parkinson's disease. It is therefore of high interest to identify molecules with trophic effects on this neuronal population. We show here that the neuregulin receptor ErbB4 is differentially expressed in mesencephalic dopaminergic neurons, found in the substantia nigra and in a subregion of the ventral tegmentum but not in the retrorubral field. Early developmental onset and continued expression of ErbB4 into the adult and the presence of two high affinity ligands, neuregulin-1 and betacellulin, in the basal ganglia, suggested that these molecules might participate in the differentiation and/or maintenance of the nigrostriatal system. In order to address this hypothesis, we used a loxP flanked ErbB4 allele in combination with a nestin-Cre transgene and generated brain-specific ErbB4 null mice. These mutant animals survived into adulthood. The distribution of dopaminergic cell bodies in the midbrain, the expression of numerous genes specific to mesencephalic dopaminergic neurons, and the axonal projection to the basal ganglia all appeared normal. Finally, an assessment of their motor function revealed no behavioral deficits. The apparent lack of any mutant phenotype suggests the presence of a strong compensatory mechanism.

Midbrain dopaminergic neurons: control of their cell fate by the engrailed transcription factors.

As for any other cell population, the development, cell fate, and properties of mesencephalic dopaminergic (mesDA) neurons are ultimately controlled at the transcriptional level. The genes for two transcription factors Engrailed-1 ( En1) and Engrailed-2 ( En2) play an essential role in the development and maintenance of these cells. They belong to a family of genes that have been investigated in Drosophila for more than half a century. The products of these genes are all characterized by homeotic tissue transformation and a highly conserved protein sequence, the homeobox. En1 and En2 act upon at least two steps of the differentiation of mesDA neurons. They take part in the regionalization event, which gives rise to the neuroepithelium that provides the precursor cells in the ventral midbrain with the fibroblast growth factor 8 signal necessary for their induction. Additionally, these genes are required in postmitotic mesDA neurons in which they are expressed from embryonic day 12 continuously into adulthood. In mutant mice homozygous null for En1 and En2, the neurons are generated in the ventral midbrain, become postmitotic, and begin to express their neurotransmitter phenotype. However, thereafter, they rapidly die by apoptosis. Cell mixing experiments in vitro and in vivo have demonstrated that the engrailed requirement for the survival of mesDA neurons is cell-autonomous. The inactivation of engrailed by RNA interference induces apoptosis in less than 24 h. These data suggest that the engrailed genes control an essential mechanism for the survival of mesDA neurons.

Engrailed genes are cell-autonomously required to prevent apoptosis in mesencephalic dopaminergic neurons.

The neuropathological hallmark of Parkinson's disease is the loss of dopaminergic neurons in the substantia nigra pars compacta, presumably mediated by apoptosis. The homeobox transcription factors engrailed 1 and engrailed 2 are expressed by this neuronal population from early in development to adulthood. Despite a large mid-hindbrain deletion in double mutants null for both genes, mesencephalic dopaminergic (mDA) neurons are induced, become postmitotic and acquire their neurotransmitter phenotype. However, at birth, no mDA neurons are left. We show that the entire population of these neurons is lost by E14 in the mutant animals, earlier than in any other described genetic model system for Parkinson's disease. This disappearance is caused by apoptosis revealed by the presence of activated caspase 3 in the dying tyrosine hydroxylase-positive mutant cells. Furthermore, using in vitro cell mixing experiments and RNA interference on primary cell culture of ventral midbrain we were able to show that the demise of mDA neurons in the mutant mice is due to a cell-autonomously requirement of the engrailed genes and not a result of the missing mid-hindbrain tissue. Gene silencing in the postmitotic neurons by RNA interference activates caspase 3 and induces apoptosis in less than 24 hours. This rapid induction of cell death in mDA neurons suggests that the engrailed genes participate directly in the regulation of apoptosis, a proposed mechanism for Parkinson's disease.

Identification and developmental analysis of genes expressed by dopaminergic neurons of the substantia nigra pars compacta.

The hallmark of Parkinson's Disease is the degenerative loss of mesencephalic dopaminergic (mDA) neurons. Previous studies have shown that the homeobox transcription factors, engrailed-1 and -2, are essential for the survival of these cells. To identify genes downstream of engrailed-1 and -2, we performed a PCR-based differential display, comparing RNA from engrailed-1/2 double mutant and wild type ventral midbrain of different embryonic ages to adult olfactory bulb, a source of unrelated DA neurons. Here, we report the result of this experiment and describe the developmental expression pattern in the ventral midbrain of three of the isolated genes, HNF3alpha, synaptotagmin I, and Ebf3. Though not regulated by engrailed-1 and -2, the expression of all three genes is limited to mDA neurons and a few other brain areas. HNF3alpha appears in the precursors of mDA neurons at E9 and is expressed in the adult brain almost exclusively by this neuronal population. Synaptotagmin I is expressed from E14 into adulthood. Ebf3, in contrast, is transiently expressed during early postmitotic differentiation.

Midbrain dopaminergic neurons: determination of their developmental fate by transcription factors.

Midbrain dopaminergic neurons are the main source of dopamine in the mammalian central nervous system and are associated with one of the most prominent human neurological disorders, Parkinson's disease. During development, they are induced in the ventral midbrain by an interaction between two diffusible factors, SHH and FGF8. The local identity of this part of the midbrain is probably determined by the combinatorial expression of three transcription factors, Otx2, Pax2, and Pax5. After the last cell division, the neurons start to express transcription factors that control further differentiation and the manifestation of cellular properties characteristic for adult dopaminergic neurons of the substantia nigra compacta and the ventral tegmentum. The first to appear is the LIM-homeodomain transcription factor, Lmx1b. It is essential for the survival of these neurons, and it regulates the expression of another transcription factor, Pitx3, an activator of tyrosine hydroxylase. Lmx1b is followed by the orphan steroid receptor Nurr1. It is essential for the expression of the dopaminergic phenotype. Several genes involved in dopamine synthesis, transport, release, and reuptake are regulated by Nurr1. This requirement is specific to the midbrain dopaminergic neurons, since other populations of the same neurotransmitter phenotype develop normally in absence of the gene. A day after Nurr1, two homeodomain transcription factors, engrailed-1 and -2, are expressed. In animals deficient in the two genes, the midbrain dopaminergic neurons are generated, but then fail to differentiate and disappear very rapidly. Interestingly, alpha-synuclein, a gene recently linked to familial forms of Parkinson's disease, is regulated by engrailed-1 and -2.