Paola Sgadò

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.

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 Parkinsons 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 Parkinsons disease and could assist in the development of novel treatments for this neurodegenerative disorder.

Midbrain Dopaminergic Neurons

Abstract: 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, Parkinsons 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 Parkinsons disease, is regulated by engrailed‐1 and ‐2.

Mutant mouse models of autism spectrum disorders.

Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental diseases characterized by a triad of specific behavioral traits: abnormal social interactions, communication deficits and stereotyped or repetitive behaviors. Several recent studies showed that ASDs have a strong genetic basis, contributing to the discovery of a number of ASD-associated genes. Due to the genetic complexity of these disorders, mouse strains with targeted deletion of ASD genes have become an essential tool to investigate the molecular and neurodevelopmental mechanisms underlying ASD. Here we will review the most relevant genetic mouse models developed by targeted inactivation of ASD-associated genes, and discuss their importance for the development of novel pharmacological therapies of these disorders.

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

BackgroundThe 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 Parkinsons disease-like molecular and behavioral deficits.ResultsIn 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.ConclusionOur study links the survival function of the Engrailed genes in developing mesDA neurons to the regulation of P75NTRand 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 P75NTRthat alter their expression levels could, in part, determine susceptibility to Parkinsons disease.

Transcriptome profiling in engrailed-2 mutant mice reveals common molecular pathways associated with autism spectrum disorders

BackgroundTranscriptome analysis has been used in autism spectrum disorder (ASD) to unravel common pathogenic pathways based on the assumption that distinct rare genetic variants or epigenetic modifications affect common biological pathways. To unravel recurrent ASD-related neuropathological mechanisms, we took advantage of the En2-/- mouse model and performed transcriptome profiling on cerebellar and hippocampal adult tissues.MethodsCerebellar and hippocampal tissue samples from three En2-/- and wild type (WT) littermate mice were assessed for differential gene expression using microarray hybridization followed by RankProd analysis. To identify functional categories overrepresented in the differentially expressed genes, we used integrated gene-network analysis, gene ontology enrichment and mouse phenotype ontology analysis. Furthermore, we performed direct enrichment analysis of ASD-associated genes from the SFARI repository in our differentially expressed genes.ResultsGiven the limited number of animals used in the study, we used permissive criteria and identified 842 differentially expressed genes in En2-/- cerebellum and 862 in the En2-/- hippocampus. Our functional analysis revealed that the molecular signature of En2-/- cerebellum and hippocampus shares convergent pathological pathways with ASD, including abnormal synaptic transmission, altered developmental processes and increased immune response. Furthermore, when directly compared to the repository of the SFARI database, our differentially expressed genes in the hippocampus showed enrichment of ASD-associated genes significantly higher than previously reported. qPCR was performed for representative genes to confirm relative transcript levels compared to those detected in microarrays.ConclusionsDespite the limited number of animals used in the study, our bioinformatic analysis indicates the En2-/- mouse is a valuable tool for investigating molecular alterations related to ASD.

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

BackgroundThe 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.ResultsHere, 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.ConclusionsThe 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.

Altered GABAergic markers, increased binocularity and reduced plasticity in the visual cortex of Engrailed-2 knockout mice

The maturation of the GABAergic system is a crucial determinant of cortical development during early postnatal life, when sensory circuits undergo a process of activity-dependent refinement. An altered excitatory/inhibitory balance has been proposed as a possible pathogenic mechanism of autism spectrum disorders (ASD). The homeobox-containing transcription factor Engrailed-2 (En2) has been associated to ASD, and En2 knockout (En2−/−) mice show ASD-like features accompanied by a partial loss of cortical GABAergic interneurons. Here we studied GABAergic markers and cortical function in En2−/− mice, by exploiting the well-known anatomical and functional features of the mouse visual system. En2 is expressed in the visual cortex at postnatal day 30 and during adulthood. When compared to age-matched En2+/+ controls, En2−/− mice showed an increased number of parvalbumin (PV+), somatostatin (SOM+), and neuropeptide Y (NPY+) positive interneurons in the visual cortex at P30, and a decreased number of SOM+ and NPY+ interneurons in the adult. At both ages, the differences in distinct interneuron populations observed between En2+/+ and En2−/− mice were layer-specific. Adult En2−/− mice displayed a normal eye-specific segregation in the retino-geniculate pathway, and in vivo electrophysiological recordings showed a normal development of basic functional properties (acuity, response latency, receptive field size) of the En2−/− primary visual cortex. However, a significant increase of binocularity was found in P30 and adult En2−/− mice, as compared to age-matched controls. Differently from what observed in En2+/+ mice, the En2−/− primary visual cortex did not respond to a brief monocular deprivation performed between P26 and P29, during the so-called “critical period.” These data suggest that altered GABAergic circuits impact baseline binocularity and plasticity in En2−/− mice, while leaving other visual functional properties unaffected.

Hippocampal Dysregulation of Neurofibromin-Dependent Pathways Is Associated with Impaired Spatial Learning in Engrailed 2 Knock-Out Mice

Genome-wide association studies indicated the homeobox-containing transcription factor Engrailed-2 (En2) as a candidate gene for autism spectrum disorders (ASD). Accordingly, En2 knock-out (En2−/−) mice show anatomical and behavioral “ASD-like” features, including decreased sociability and learning deficits. The molecular pathways underlying these deficits in En2−/− mice are not known. Deficits in signaling pathways involving neurofibromin and extracellular-regulated kinase (ERK) have been associated with impaired learning. Here we investigated the neurofibromin-ERK cascade in the hippocampus of wild-type (WT) and En2−/− mice before and after spatial learning testing. When compared with WT littermates, En2−/− mice showed impaired performance in the Morris water maze (MWM), which was accompanied by lower expression of the activity-dependent gene Arc. Quantitative RT-PCR, immunoblotting, and immunohistochemistry experiments showed a marked downregulation of neurofibromin expression in the dentate gyrus of both naive and MWM-treated En2−/− mice. ERK phosphorylation, known to be induced in the presence of neurofibromin deficiency, was increased in the dentate gyrus of En2−/− mice after MWM. Treatment of En2−/− mice with lovastatin, an indirect inhibitor of ERK phosphorylation, markedly reduced ERK phosphorylation in the dentate gyrus, but was unable to rescue learning deficits in MWM-trained mutant mice. Further investigation is needed to unravel the complex molecular mechanisms linking dysregulation of neurofibromin-dependent pathways to spatial learning deficits in the En2 mouse model of ASD.

Reduced phosphorylation of synapsin I in the hippocampus of Engrailed-2 knockout mice, a model for autism spectrum disorders

Mice lacking the homeodomain transcription factor Engrailed-2 (En2(-/-) mice) are a well-characterized model for autism spectrum disorders (ASD). En2(-/-) mice present molecular, neuropathological and behavioral deficits related to ASD, including down-regulation of ASD-associated genes, cerebellar hypoplasia, interneuron loss, enhanced seizure susceptibility, decreased sociability and impaired cognition. Specifically, impaired spatial learning in the Morris water maze (MWM) is associated with reduced expression of neurofibromin and increased phosphorylation of extracellular-regulated kinase (ERK) in the hippocampus of En2(-/-) adult mice. In the attempt to better understand the molecular cascades underlying neurofibromin-dependent cognitive deficits in En2 mutant mice, we investigated the expression and phosphorylation of synapsin I (SynI; a major target of neurofibromin-dependent signaling) in the hippocampus of wild-type (WT) and En2(-/-) mice before and after MWM. Here we show that SynI mRNA and protein levels are down-regulated in the hippocampus of naïve and MWM-treated En2(-/-) mice, as compared to WT controls. This down-regulation is paralleled by reduced levels of SynI phosphorylation at Ser549 and Ser553 residues in the hilus of mutant mice, before and after MWM. These data indicate that in En2(-/-) hippocampus, neurofibromin-dependent pathways converging on SynI phosphorylation might underlie hippocampal-dependent learning deficits observed in En2(-/-) mice.