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.dopamine ͉ midbrain ͉ mouse mutant ͉ neurodegenerative disease ͉ substantia nigra M esencephalic dopaminergic (mesDA) neurons are the main source of dopamine in the mammalian central nervous system (1). They are located in three distinct nuclei, the substantia nigra pars compacta (SN), the ventral tegmentum (VTA), and the retrorubral field (RRF). Their main innervation targets are the basal ganglia, where they play a major role in controlling emotion, motivation, and motor behavior (2, 3), recognized by their involvement in schizophrenia, addiction, and most prominently in Parkinson's disease (PD; ref. 4). PD is a multisystemic degenerative disorder of the central and peripheral nervous systems. Its hallmark is the progressive degeneration of DA neurons in the SN. The clinical symptoms are resting tremor; muscular rigidity; postural instability; hyposomia; a positive response to the application of L-3,4-dihydroxyphenylalanine (L-DOPA); and the presence of cytoplasmatic inclusions (Lewy bodies) in postmortem brains (5). Additionally, PD patients are often characterized by weight loss starting before diagnosis and continuing with disease progression (6, 7).The homeodomain transcription factors Engrailed-1 and -2 (En1 and En2) are expressed by all mesDA neurons soon after differentiation and then continuously into adulthood. They are cell-autonomously and in a gene-dose-dependent manner required for the survival of this neuronal population, leading to the complete loss of the cells in mutant mice homozygous null for both genes (En1Ϫ͞Ϫ;En2Ϫ͞Ϫ; refs. 8 and 9). In their function as survival factors for mesDA neurons, the two genes are redundant and can compensate for each other (9, 10). The single-null mutants for either En1 (En1Ϫ͞Ϫ) or En2 (En2Ϫ͞Ϫ) show no significant alterations in the organization of the mesDA system at birth, but the mice with genotypes intermediate to the single and double mutants are distinctively different from each other. Whereas En1Ϫ͞Ϫ;En2ϩ͞Ϫ mice die at po...

In humans, inclusion or exclusion of the fibronectin EDA exon is mainly regulated by a polypurinic enhancer element (exonic splicing enhancer [ESE]) and a nearby silencer element (exonic splicing silencer [ESS]).While human and mouse ESEs behave identically, mutations introduced into the homologous mouse ESS sequence result either in no change in splicing efficiency or in complete exclusion of the exon. Here, we show that this apparently contradictory behavior cannot be simply accounted for by a localized sequence variation between the two species. Rather, the nucleotide differences as a whole determine several changes in the respective RNA secondary structures. By comparing how the two different structures respond to homologous deletions in their putative ESS sequences, we show that changes in splicing behavior can be accounted for by a differential ESE display in the two RNAs. This is confirmed by RNA-protein interaction analysis of levels of SR protein binding to each exon. The immunoprecipitation patterns show the presence of complex multi-SR protein-RNA interactions that are lost with secondary-structure variations after the introduction of ESE and ESS variations. Taken together, our results demonstrate that the sequence context, in addition to the primary sequence identity, can heavily contribute to the making of functional units capable of influencing pre-mRNA splicing.The splicing process is a very flexible and critical step in gene expression. In fact, selected removal or inclusion of individual exons from nascent mRNA molecules allows a single gene to generate multiple proteins with different primary structures; in these cases, the process is known as alternative splicing (1, 38). Constitutive and alternative splicing processes are catalyzed by the spliceosome, a very complex RNA-protein aggregate that has been recently estimated to contain approximately 145 different proteins in addition to the five spliceosomal snRNAs (1,38,41,68). These factors are responsible for accurate positioning of the spliceosome on the 5Ј and 3Ј splice sequences that define the exon. However, correct positioning of the spliceosome is a very complex process owing to the degeneracy of the splice site consensus sequences, the presence of cryptic splice sites in large introns, and the fact that most pre-mRNAs contain multiple introns (26). Therefore, the action of several different proteins is required to achieve accurate positioning of the spliceosome on the splice site. Not surprisingly, alterations in the splicing process have been increasingly reported as being involved in many genetic diseases (5,8,13,23,58). Among the well-known factors that may heavily influence the identification of intron-exon boundaries by the spliceosome are the exon length (3, 65), the presence of splicing enhancer and silencer elements (5, 38), the strength of splicing signals (26), and the promoter architecture (19,33). In addition to these factors, it has been proposed that the natural tendency of RNAs to fold in highly stable secondary and tertia...

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 α-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.

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, α‐synuclein, a gene recently linked to familial forms of Parkinson's disease, is regulated by engrailed‐1 and ‐2.

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