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Background: PTEN Hamartoma Tumor Syndrome (PHTS) is characterized by frequent mutation in PTEN gene. PHTS patients have numerous neurological defects including macrocephaly and autism spectrum disorder. While clinical features of PHTS are likely the result of PTEN haploinsufficiency, the role of PTEN gene dosage in driving transcriptomic changes in specific neural cell types is not known. Methods: We performed transcriptomic and proteomic analysis on neural tissues and primary cultures from Pten heterozygous and homozygous knockout mice. Results: We found that somatosensory cortex (SSC) of Pten heterozygous mice are enriched with immune response and oligodendrocyte development Gene Ontology (GO) terms. Parallel proteomic analysis reveals differentially expressed proteins (DEPs) related to dendritic spine development, keratinization and hamartoma signatures. The lack of concordance between transcriptomic and proteomic data prompted us to profile the transcriptomes of specific neural cell types. Overall, Pten homozygous knockout cells have much greater differential expressed genes (DEGs) than Pten heterozygous knockout cells. Pten heterozygous knockout primary astrocytes (AST) are enriched in Extracellular Matrix GO terms while primary cortical neurons (PCN) are enriched with immediate-early genes. In Pten homozygous knockout AST, cilium-related activity is enriched while PCN exhibited downregulation of forebrain neuron generation and differentiation which may lead to disruption of excitatory/inhibitory balance in brain. By integrating DEPs with a pre-filtered DEGs, we identified traits of intelligence and cognitive function, and schizophrenia being enriched, and DEP from AST is significantly associated with intelligence and depression.Limitations: First, bulk tissues were used for transcriptomic and proteomics analysis. Cell type specific expression changes may not be revealed. Second, primary neural cell cultures were used for transcriptomic analysis, which may not reflect the normal physiological conditions.Conclusions: We have uncovered transcriptomic alterations and molecular pathways that may explain neurological disorders associated with PHTS.

Neurodevelopmental and neuropsychiatric disorders, such as autism spectrum disorders (ASD), anorexia nervosa (AN), Alzheimer’s disease (AD), and schizophrenia (SZ), are heterogeneous brain disorders with unknown etiology. Genome wide studies have revealed a wide variety of risk genes for these disorders, indicating a biological link between genetic signaling pathways and brain pathology. A unique risk gene is Contactin 4 (Cntn4), an Ig cell adhesion molecule (IgCAM) gene, which has been associated with several neuropsychiatric disorders including ASD, AN, AD, and SZ. Here, we investigated the Cntn4 gene knockout (KO) mouse model to determine whether memory dysfunction and altered brain plasticity, common neuropsychiatric symptoms, are affected by Cntn4 genetic disruption. For that purpose, we tested if Cntn4 genetic disruption affects CA1 synaptic transmission and the ability to induce LTP in hippocampal slices. Stimulation in CA1 striatum radiatum significantly decreased synaptic potentiation in slices of Cntn4 KO mice. Neuroanatomical analyses showed abnormal dendritic arborization and spines of hippocampal CA1 neurons. Short- and long-term recognition memory, spatial memory, and fear conditioning responses were also assessed. These behavioral studies showed increased contextual fear conditioning in heterozygous and homozygous KO mice, quantified by a gene-dose dependent increase in freezing response. In comparison to wild-type mice, Cntn4-deficient animals froze significantly longer and groomed more, indicative of increased stress responsiveness under these test conditions. Our electrophysiological, neuro-anatomical, and behavioral results in Cntn4 KO mice suggest that Cntn4 has important functions related to fear memory possibly in association with the neuronal morphological and synaptic plasticity changes in hippocampus CA1 neurons.

Autism spectrum disorder (ASD) is characterized by impaired social interaction, language delay and repetitive or restrictive behaviors. With increasing prevalence, ASD is currently estimated to affect 0.5–2.0% of the global population. However, its etiology remains unclear due to high genetic and phenotypic heterogeneity. Copy number variations (CNVs) are implicated in several forms of syndromic ASD and have been demonstrated to contribute toward ASD development by altering gene dosage and expression. Increasing evidence points toward the p-arm of chromosome 3 (chromosome 3p) as an ASD risk locus. Deletions occurring at chromosome 3p result in 3p-deletion syndrome (Del3p), a rare genetic disorder characterized by developmental delay, intellectual disability, facial dysmorphisms and often, ASD or ASD-associated behaviors. Therefore, we hypothesize that overlapping molecular mechanisms underlie the pathogenesis of Del3p and ASD. To investigate which genes encoded in chromosome 3p could contribute toward Del3p and ASD, we performed a comprehensive literature review and collated reports investigating the phenotypes of individuals with chromosome 3p CNVs. We observe that high frequencies of CNVs occur in the 3p26.3 region, the terminal cytoband of chromosome 3p. This suggests that CNVs disrupting genes encoded within the 3p26.3 region are likely to contribute toward the neurodevelopmental phenotypes observed in individuals affected by Del3p. The 3p26.3 region contains three consecutive genes encoding closely related neuronal immunoglobulin cell adhesion molecules (IgCAMs): Close Homolog of L1 (CHL1), Contactin-6 (CNTN6), and Contactin-4 (CNTN4). CNVs disrupting these neuronal IgCAMs may contribute toward ASD phenotypes as they have been associated with key roles in neurodevelopment. CHL1, CNTN6, and CNTN4 have been observed to promote neurogenesis and neuronal survival, and regulate neuritogenesis and synaptic function. Furthermore, there is evidence that these neuronal IgCAMs possess overlapping interactomes and participate in common signaling pathways regulating axon guidance. Notably, mouse models deficient for these neuronal IgCAMs do not display strong deficits in axonal migration or behavioral phenotypes, which is in contrast to the pronounced defects in neuritogenesis and axon guidance observed in vitro. This suggests that when CHL1, CNTN6, or CNTN4 function is disrupted by CNVs, other neuronal IgCAMs may suppress behavioral phenotypes by compensating for the loss of function.

The nervous system functions by virtue of precisely positioned neurons with fine-tuned circuitry. The formation of these structures depends largely on interactions and communication between different cells mediated by proteins on the extracellular membrane. Proteins carrying the immunoglobulin amino acid motif are manifold and encountered in all species, organ systems and cell-types, where they are engaged in many biological processes. About eight brain-expressed subclasses combine the Ig motif with fibronectin type-3 (FNIII) domains and form the FN-type IgCAMs, in total consisting of over a hundred distinct protein (iso)forms. In the developing nervous system, they serve as cell adhesion molecules, (co)receptors, ligands, tyrosine kinases and phosphatases. These activities are recognized in the involvement of FN-type IgCAMs in neurodevelopmental processes as neurite outgrowth, axon elongation and steering, neuronal and glial migration, neuron–glia interaction, synaptogenesis and synaptic plasticity. In accord with these elementary neurodevelopmental functions, mutations in genes encoding these FN-type IgCAMs have been encountered in several neurodevelopmental disorders and syndromes, including mental retardation and autism.

FMRFamide is a neuropeptide that is widely distributed in invertebrates and known to be involved in many physiological functions. Previously we noted marked differences in expression of thefmrfgene in the stellate ganglion ofDoryteuthis pealei*compared to the central nervous system. In this study we aimed to examen the brain systems ofDoryteuthis pealei*for the presence and distribution offmrf-expressing cells and fiber networks. Late squid embryos and hatchlings were examined by in situ hybridization and immunohistochemistry in whole mounts and tissue sections. All central lobes contained limited numbers of scattered neurons expressingfmrf, but the FMRFamide-containing fiber systems were abundant and extensive, mostly present in the neuropil of lobes. Main clusters of neurons were located in the magnocellular and chromatophore lobes of the posterior subesophageal mass (PSM), and in dorsal aspects of the basal lobe (BL). Dense FMRFamide-immunoreactive fibers were particularly seen in the optic lobe (OL), medial and posterior supraesophageal masses (MSM and SPM) often with a commissural organization. The data show that the central lobes ofDoryteuthis pealeihatchlings have a matured FMRFamide system organized in a limited number of centers, but with widely distributed efferents. This suggests that FMRFamide neurons are already functionally engaged in the late embryo. The localization indicates that control of chromatophores and fin movement are amongst these functions.

Background Animal models for neurodevelopmental disorders (NDD) generally rely on a single genetic mutation on a fixed genetic background. Recent human genetic studies however indicate that a clinical diagnosis with ASDAutism Spectrum Disorder (ASD) is almost always associated with multiple genetic fore- and background changes. The translational value of animal model studies would be greatly enhanced if genetic insults could be studied in a more quantitative framework across genetic backgrounds. Methods We used the Collaborative Cross (CC), a novel mouse genetic reference population, to investigate the quantitative genetic architecture of mouse behavioral phenotypes commonly used in animal models for NDD. Results Classical tests of social recognition and grooming phenotypes appeared insufficient for quantitative studies due to genetic dilution and limited heritability. In contrast, digging, locomotor activity, and stereotyped exploratory patterns were characterized by continuous distribution across our CC sample and also mapped to quantitative trait loci containing genes associated with corresponding phenotypes in human populations. Conclusions These findings show that the CC can move animal model studies beyond comparative single gene-single background designs, and point out which type of behavioral phenotypes are most suitable to quantify the effect of developmental etiologies across multiple genetic backgrounds. Electronic supplementary material The online version of this article (10.1186/s13229-018-0252-2) contains supplementary material, which is available to authorized users.

Morphological screening of mouse brains with known behavioral deficits can give great insight into the relationship between brain regions and their behavior. Oxytocin- and CD38-deficient mice have previously been shown to have behavioral phenotypes, such as restrictions in social memory, social interactions, and maternal behavior. CD38 is reported as an autism spectrum disorder (ASD), and candidate gene and these behavioral phenotypes may be linked to ASD. To address whether these behavioral phenotypes relate to brain pathology and neuronal morphology, here we investigate the morphological changes in the CD38-deficient mice brains, with focus on the pathology and neuronal morphology of the cortex and hippocampus, using Nissl staining, immunohistochemistry, and Golgi staining. No difference was found in terms of cortical layer thickness. However, we found abnormalities in the number of neurons and neuron morphology in the visual cortex and dentate gyrus (DG). In particular, there were arborisation differences between CD38-/- and CD38+/+ mice in the apical dendrites of the visual cortex and hippocampal CA1 pyramidal neurons. The data suggest that CD38 is implicated in appropriate development of brain regions important for social behavior.

Contactin-5 (Cntn5) is an immunoglobulin cell adhesion molecule that is exclusively expressed in the central nervous system. In view of its association with neurodevelopmental disorders, particularly autism spectrum disorder (ASD), this study focused on Cntn5-positive areas in the forebrain and aimed to explore the morphological and behavioral phenotypes of the Cntn5 null mutant (Cntn5−/−) mouse in relation to these areas and ASD symptomatology. A newly generated antibody enabled us to elaborately describe the spatial expression pattern of Cntn5 in P7 wild type (Cntn5+/+) mice. The Cntn5 expression pattern included strong expression in the cerebral cortex, hippocampus and mammillary bodies in addition to described previously brain nuclei of the auditory pathway and the dorsal thalamus. Thinning of the primary somatosensory (S1) cortex was found in Cntn5−/− mice and ascribed to a misplacement of Cntn5-ablated cells. This phenotype was accompanied by a reduction in the barrel/septa ratio of the S1 barrel field. The structure and morphology of the hippocampus was intact in Cntn5−/− mice. A set of behavioral experiments including social, exploratory and repetitive behaviors showed that these were unaffected in Cntn5−/− mice. Taken together, these data demonstrate a selective role of Cntn5 in development of the cerebral cortex without overt behavioral phenotypes.

Astrocytes contribute to the formation, function, and plasticity of synapses. Their processes enwrap the neuronal components of the tripartite synapse, and due to this close interaction they are perfectly positioned to modulate neuronal communication. The interaction between astrocytes and synapses is facilitated by cell adhesion molecules and matricellular proteins, which have been implicated in the formation and functioning of tripartite synapses. The importance of such neuron-astrocyte integration at the synapse is underscored by the emerging role of astrocyte dysfunction in synaptic pathologies such as autism and schizophrenia. Here we review astrocyte-expressed cell adhesion molecules and matricellular molecules that play a role in integration of neurons and astrocytes within the tripartite synapse.

FoxK2 is a forkhead transcription factor expressed ubiquitously in the developing murine central nervous system. Here we investigated the role of FoxK2 in vitro and focused on proliferation and cellular survival. Knockdown of FoxK2 results in a decrease in BrdU incorporation and H3 phosphorylation, suggesting attenuation of proliferation. In the absence of growth factors FoxK2 knockdown results in a dramatic increase in caspase 3 activity and propidium iodide positive cells, indicative of cell death. Additionally, knockdown of FoxK2 results in an increase in the mRNA of Gadd45α, Gadd45γ, as well as an increase in the phosphorylation of the mTOR dependent kinase p70S6K. Rapamycin treatment completely blocked the increase in p70S6K and synergistically potentiated the decrease in H3 phosphorylation upon FoxK2 knockdown. To gain more insight into the pro-apoptotic effects upon FoxK2 knockdown we screened for changes in Bcl2 genes. Upon FoxK2 knockdown both Puma and Noxa were significantly upregulated. Both genes were not inhibited by rapamycin treatment, instead rapamycin increased Noxa mRNA. FoxK2 requirement in cellular survival is further emphasized by the fact that resistance to TGFβ-induced cell death was greatly diminished after FoxK2 knockdown. Overall our data suggest FoxK2 is required for proliferation and survival, that mTOR is part of a feedback loop partly compensating for FoxK2 loss, possibly by upregulating Gadd45s, whereas cell death upon FoxK2 loss is induced in a Bcl2 dependent manner via Puma and Noxa.

Summary The giant fiber system of the squid Loligo pealei mediates the escape response and is an important neurobiological model. Here, we identified an abundant transcript in the stellate ganglion (SG) that encodes a FMRFamide precursor, and characterized FMRFamide and FI/LRF-amide peptides. To determine whether FMRFamide plays a role in the adult and hatchling giant fiber system, we studied the expression of the Fmrf gene and FMRFamide peptides. In stage 29 embryos and stage 30 hatchlings, Ffmr transcripts and FMRFamide peptide were low to undetectable in the SG, in contrast to groups of neurons intensely expressing the Fmrf gene in several brain lobes, including those that innervate the SG. In the adult SG the Fmrf gene was highly expressed, but the FMRFamide peptide was in low abundance. Intense staining for FMRFamide in the adult SG was confined to microneurons and fibers in the neuropil and to small fibers surrounding giant axons in stellar nerves. This shows that the Fmrf gene in the SG is strongly regulated post-hatching, and suggests that the FMRFamide precursor is incompletely processed in the adult SG. The data suggest that the SG only employs the Fmrf gene post-hatching and restricts the biosynthesis of FMRFamide, demonstrating that this peptide is not a major transmitter of the giant fiber system. This contrasts with brain lobes that engage FMRFamide embryonically as a regulatory peptide in multiple neuronal systems, including the afferent fibers that innervate the SG. The biological significance of these mechanisms may be to generate diversity within Fmrf-expressing systems in cephalopods.

Autism is a disease of brain plasticity. Inspiring work of Willem Hendrik Gispen on neuronal plasticity has stimulated us to investigate gene defects in autism and the consequences for brain development. The central process in the pathogenesis of autism is local dendritic mRNA translation which is dependent on axodendritic communication. Hence, most autism-related gene products (i) are part of the protein synthesis machinery itself, (ii) are components of the mTOR signal transduction pathway, or (iii) shape synaptic activity and plasticity. Accordingly, prototype drugs have been recognized that interfere with these pathways. The contactin (CNTN) family of Ig cell adhesion molecules (IgCAMs) harbours at least three members that have genetically been implicated in autism: CNTN4, CNTN5, and CNTN6. In this chapter we review the genetic and neurobiological data underpinning their role in normal and abnormal development of brain systems, and the consequences for behavior. Although data on each of these CNTNs are far from complete, we tentatively conclude that these three contactins play roles in brain development in a critical phase of establishing brain systems and their plasticity. They modulate neuronal activities, such as neurite outgrowth, synaptogenesis, survival, guidance of projections and terminal branching of axons in forming neural circuits. Current research on these CNTNs concentrate on the neurobiological mechanism of their developmental functions. A future task will be to establish if proposed pharmacological strategies to counteract ASD-related symptomes can also be applied to reversal of phenotypes caused by genetic defects in these CNTN genes.

Background Mouse models offer an essential tool to unravel the impact of genetic mutations on autism-related phenotypes. The behavioral impact of some important candidate gene models for autism spectrum disorder (ASD) has not yet been studied, and existing characterizations mostly describe behavioral phenotypes at adult ages, disregarding the developmental nature of the disorder. In this context, the behavioral influence of CNTN4, one of the strongest suggested ASD candidate genes, is unknown. Here, we used our recently established developmental test battery to characterize the consequences of disruption of contactin 4 (Cntn4) on neurological, sensory, cognitive, and behavioral phenotypes across different developmental stages. Methods C57BL/6J mice with heterozygous and homozygous disruption of Cntn4 were studied through an extensive, partially longitudinal, test battery at various developmental stages, including various paradigms testing social and restricted repetitive behaviors. Results Developmental neurological and cognitive screenings revealed no significant differences between genotypes, and ASD-related behavioral domains were also unchanged in Cntn4-deficient versus wild-type mice. The impact of Cntn4-deficiency was found to be limited to increased startle responsiveness following auditory stimuli of different high amplitudes in heterozygous and homozygous Cntn4-deficient mice and enhanced acquisition in a spatial learning task in homozygous mice. Conclusions Disruption of Cntn4 in the C57BL/6J background does not affect specific autism-related phenotypes in developing or adult mice but causes subtle non-disorder specific changes in sensory behavioral responses and cognitive performance. Electronic supplementary material The online version of this article (doi:10.1186/s11689-016-9140-2) contains supplementary material, which is available to authorized users.

In view of important neurobiological functions of the cell adhesion molecule contactin-6 (Cntn6) that have emerged from studies on null-mutant mice and autism spectrum disorders (ASD) patients, we set out to examine pathways underlying functions of Cntn6 using a proteomics approach. We identified the cell adhesion GPCR latrophilin-1 (Lphn1, a.k.a. CIRL1/CL, ADGRL1) as a binding partner for Cntn6 forming together a heteromeric cis-complex. Lphn1 expression in cultured neurons caused reduction in neurite outgrowth and increase in apoptosis, which was rescued by coexpression of Cntn6. In cultured neurons derived from Cntn6-/- mice, Lphn1 knockdown reduced apoptosis, suggesting that the observed apoptosis was Lphn1-dependent. In line with these data, the number of apoptotic cells was increased in the cortex of Cntn6-/- mice compared to wild-type littermate controls. These results show that Cntn6 can modulate the activity of Lphn1 by direct binding and suggests that Cntn6 may prevent apoptosis thereby impinging on neurodevelopment.

Development of meso-diencephalic dopamine (mdDA) neurons requires the combined actions of the orphan nuclear receptor Nurr1 and the paired-like homeobox transcription factor Pitx3. Whereas all mdDA neurons require Nurr1 for expression of Th and survival, dependence on Pitx3 is displayed only by the mdDA subpopulation that will form the substantia nigra (SNc). Previously, we have demonstrated that Pitx3–/– embryos lack the expression of the retinoic acid (RA)-generating enzyme Ahd2, which is normally selectively expressed in the Pitx3-dependent DA neurons of the SNc. Restoring RA signaling in Pitx3–/– embryos revealed a selective dependence of SNc neurons on the presence of RA for differentiation into Th-positive neurons and maintenance throughout embryonic development. Whereas these data are suggestive of an important developmental role for RA in neurons of the SNc, it remained unclear whether other Nurr1 and Pitx3 target genes depend on RA signaling in a manner similar to Th. In the search for genes that were affected in Pitx3-deficient mdDA neurons and restored upon embryonic RA treatment, we provide evidence that Delta-like 1, D2R (Drd2) and Th are regulated by Pitx3 and RA signaling, which influences the mdDA terminal differentiated phenotype. Furthermore, we show that regulation of Ahd2-mediated RA signaling represents only one aspect of the Pitx3 downstream cascade, as Vmat2, Dat, Ahd2 (Aldh1a1), En1, En2 and Cck were unaffected by RA treatment and are (subset) specifically modulated by Pitx3. In conclusion, our data reveal several RA-dependent and -independent aspects of the Pitx3-regulated gene cascade, suggesting that Pitx3 acts on multiple levels in the molecular subset-specification of mdDA neurons.

Contains fulltext : 98353.pdf (Publisher’s version ) (Open Access) Recent array-based studies have detected a wealth of copy number variations (CNVs) in patients with autism spectrum disorders (ASD). Since CNVs also occur in healthy individuals, their contributions to the patient's phenotype remain largely unclear. In a cohort of children with symptoms of ASD, diagnosis of the index patient using ADOS-G and ADI-R was performed, and the Social Responsiveness Scale (SRS) was administered to the index patients, both parents, and all available siblings. CNVs were identified using SNP arrays and confirmed by FISH or array CGH. To evaluate the clinical significance of CNVs, we analyzed three families with multiple affected children (multiplex) and six families with a single affected child (simplex) in which at least one child carried a CNV with a brain-transcribed gene. CNVs containing genes that participate in pathways previously implicated in ASD, such as the phosphoinositol signaling pathway (PIK3CA, GIRDIN), contactin-based networks of cell communication (CNTN6), and microcephalin (MCPH1) were found not to co-segregate with ASD phenotypes. In one family, a loss of CNTN5 co-segregated with disease. This indicates that most CNVs may by themselves not be sufficient to cause ASD, but still may contribute to the phenotype by additive or epistatic interactions with inherited (transmitted) mutations or non-genetic factors. Our study extends the scope of genome-wide CNV profiling beyond de novo CNVs in sporadic patients and may aid in uncovering missing heritability in genome-wide screening studies of complex psychiatric disorders. 01 november 2011

In recent years, the meso-diencephalic dopaminergic (mdDA) neurons have been extensively studied for their association with Parkinson's disease. Thus far, specification of the dopaminergic phenotype of mdDA neurons is largely attributed to the orphan nuclear receptor Nurr1. In this study, we provide evidence for extensive interplay between Nurr1 and the homeobox transcription factor Pitx3 in vivo. Both Nurr1 and Pitx3 interact with the co-repressor PSF and occupy the promoters of Nurr1 target genes in concert. Moreover, in vivo expression analysis reveals that Nurr1 alone is not sufficient to drive the dopaminergic phenotype in mdDA neurons but requires Pitx3 for full activation of target gene expression. In the absence of Pitx3, Nurr1 is kept in a repressed state through interaction with the co-repressor SMRT. Highly resembling the effect of ligand activation of nuclear receptors, recruitment of Pitx3 modulates the Nurr1 transcriptional complex by decreasing the interaction with SMRT, which acts through HDACs to keep promoters in a repressed deacetylated state. Indeed, interference with HDAC-mediated repression in Pitx3-/- embryos efficiently reactivates the expression of Nurr1 target genes, bypassing the necessity for Pitx3. These data position Pitx3 as an essential potentiator of Nurr1 in specifying the dopaminergic phenotype, providing novel insights into mechanisms underlying development of mdDA neurons in vivo, and the programming of stem cells as a future cell replacement therapy for Parkinson's disease.

Summary Background Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the selective death of motor neurons in the brain and spinal cord. Genetic risk factors have been implicated in susceptibility to ALS. Like single nucleotide polymorphisms (SNPs), copy-number variants (CNVs) are a source of genetic variation that have important effects on gene expression and disease phenotypes, and our aim was to identify CNVs that predispose to sporadic ALS. Methods We did a genome-wide screen for CNVs by analysis of Illumina 317K SNP arrays for 406 patients with sporadic ALS and 404 controls. We examined CNVs for association with ALS, and used the Kyoto Encyclopedia of Genes and Genomes database and the Gene Ontology database to investigate the functionality of genes that were deleted exclusively in patients with ALS. Findings We detected 2328 CNVs in 810 individuals. No CNV locus was significantly associated with sporadic ALS. 406 genes were duplicated or deleted exclusively in patients with ALS and have not been reported in previous studies of CNVs. Of the 390 genes heterozygously deleted in patients with sporadic ALS, 155 (40%) deletions were recorded exclusively in patients. By contrast, of the 323 genes heterozygously deleted in control participants, only 51 (16%) were exclusive to the controls (p=2·15×10 −12 for difference between groups). Products of the genes deleted specifically in patients with sporadic ALS include proteins involved in oxidative phosphorylation, regulation of the actin cytoskeleton, and interactions between cytokines and their receptors. Interpretation Common CNVs in the regions of the genome represented on the SNP array are unlikely to be associated with sporadic ALS. However, the high number of genes deleted specifically in patients with ALS strongly suggests that multiple rare deletions might have an important role in ALS pathogenesis.

Selective neuronal loss in the substantia nigra (SNc), as described for Parkinson's 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.

Dopaminergic neurons located in the ventral mesodiencephalon are essential for the control of voluntary movement and the regulation of emotion, and are severely affected in neurodegenerative diseases such as Parkinson's disease. Recent advances in molecular biology and mouse genetics have helped to unravel the mechanisms involved in the development of mesodiencephalic dopaminergic (mdDA) neurons, including their specification, migration and differentiation, as well as the processes that govern axonal pathfinding and their specific patterns of connectivity and maintenance. Here, we follow the developmental path of these neurons with the goal of generating a molecular code that could be exploited in cell-replacement strategies to treat diseases such as Parkinson's disease.

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 murine forkhead family of transcription factors consists of over 30 members, the vast majority of which is important in embryonic development. Implicated in processes such as proliferation, differentiation and survival, forkhead factors show highly restricted expression patterns. In search for forkhead genes expressed in specific neural systems, we identified multiple family members. We performed a detailed expression analysis for Foxj2, Foxk1 and the murine orthologue of the human ILF1 gene, which show a remarkable preference for complex cortical structures. In addition, a comprehensive examination of forkhead gene expression in dopamine neurons of the ventral tegmental area and substantia nigra pars compacta, revealed Ilf1 as a novel transcriptional regulator in midbrain dopamine neurons. These forkhead transcription factors may play a role in maintenance and survival of developing and adult neurons.

Specific vulnerability of substantia nigra compacta neurons as compared to ventral tegmental area neurons, as emphasized in Parkinson's disease, has been studied for many years and is still not well understood. The molecular codes and mechanisms that drive development of these structures have recently been studied through the use of elegant genetic ablation experiments. The data suggested that specific genes at specific anatomical positions in the ventricular zone are crucial to drive development of young neurons into the direction of the dopaminergic phenotype. In addition, it has become clear the these dopaminergic neurons are present in the diencephalon and in the mesencephalon and that they may contain a specific molecular signature that defines specific subsets in terms of position and function. The data indicate that these specific subsets may explain the specific response of these neurons to toxins and genetic ablation.

Parkinson's 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.

Forkhead members of the ‘O’ class (FoxO) are transcription factors crucial for the regulation of metabolism, cell cycle, cell death and cell survival. FoxO factors are regulated by insulin-mediated activation of PI3K (phosphoinositide 3-kinase)–PKB (protein kinase B) signalling. Activation of PI3K–PKB signalling results in the phosphorylation of FoxO factors on three conserved phosphorylation motifs, which are essential for the translocation of FoxO factors from the nucleus to the cytosol. FoxO6, however, remains mostly nuclear due to the fact that its shuttling ability is dramatically impaired. FoxO1, FoxO3 and FoxO4 all contain an N- and C-terminal PKB motif and a motif located in the forkhead domain. FoxO6 lacks the conserved C-terminal PKB motif, which is the cause of the shuttling impairment. Since FoxO6 can be considered constitutively nuclear, we investigated whether it is also a constitutively active transcription factor. Our results show that FoxO6 transcriptional activity is inhibited by growth factors, independent of shuttling, indicating that it is not constitutively active. The PKB site in the forkhead domain (Ser184) regulated the DNA binding characteristics and the N-terminal PKB site acted as a growth factor sensor. In summary, FoxO6 is not a constitutively active transcription factor and can be regulated by growth factors in a Thr26- and Ser184-dependent manner, independent of shuttling to the cytosol.

Item does not contain fulltext BACKGROUND INFORMATION: Many neurons secrete classical transmitters from synaptic vesicles as well as peptide transmitters from LDCVs (large dense-core vesicles). Little is known about the mechanistic differences between these two secretory pathways. The soluble protein Munc18-1 is essential for synaptic vesicle secretion [Verhage, Maia, Plomp, Brussaard, Heeroma, Vermeer, Toonen, Hammer, van den Berg, Missler, et al. (2000) Science 287, 864-869.]. RESULTS: In the present study, we tested if Munc18 genes are also involved in peptidergic secretion from LDCVs using the anterior pituitary as a model system. We show that Munc18-1 is the dominant isoform expressed in the anterior pituitary. In Munc18-1 null mutant mice, the anterior pituitary developed normally and the five major endocrine cell types had a normal distribution. However, circulating peptide hormone levels were decreased by up to 50-fold in the null mutant, whereas the intracellular levels were significantly higher than that in controls. Ultrastructural analysis using the tannic acid method revealed striking differences in the distribution of secretory vesicles: (i) the number of exocytotic figures was mostly decreased in the null mutants and (ii) the LDCVs accumulated near but not at their target membrane. This is in contrast with the apparently normal distribution of synaptic vesicles in developing synapses in the null mutant (Verhage et al., 2000). CONCLUSIONS: We conclude that Munc18-1 is involved in the secretion of peptide hormones and in the docking of LDCVs. These results unmask an apparent mechanistic difference between LDCVs and synaptic vesicles.

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.

Although mu-opioid receptors have been extensively investigated for their role in drug reinforcement, little is known about the contribution of these receptors to the acute and sensitized locomotor response to cocaine. In this study mu-opioid receptor involvement in acute cocaine-induced locomotor activity and in the development of cocaine-induced behavioral sensitization was evaluated using mu-opioid receptor knockout mice and chronic naltrexone (NTX) pretreatment as models. In addition, co-administration of the specific mu-opioid receptor antagonist CTOP with repeated saline or cocaine injections was used to establish the involvement of mu-opioid receptors in sensitization to the locomotor stimulant effects of cocaine. The acute locomotor response to cocaine (3, 10, 20, or 30 mg/kg i.p.) of mu-opioid receptor knockout or chronic NTX pretreated mice was not different from the cocaine response of their respective controls. With respect to cocaine-induced behavioral sensitization, induced by daily injections of 20 mg/kg cocaine for 11 subsequent days, mu-opioid receptor knockout mice developed behavioral sensitization to the locomotor stimulant effects of cocaine (challenge 10 mg/kg i.p.) comparable to wild-type littermates and the mu-opioid receptor antagonist CTOP did not affect cocaine-induced sensitization either. However, mice that were pretreated with NTX exhibited augmented cocaine-induced behavioral sensitization relative to placebo pretreated controls, which may be ascribed to increased delta-opioid receptor levels as has been described for chronic NTX pretreated mice. The present findings suggest that mu-opioid receptors are not required for the acute locomotor response to cocaine nor are they essential for the development of cocaine-induced behavioral sensitization.

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.

Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental disorders with shared symptoms in the area of communication and language, restricted interests, and stereotyped and social behaviors. Causes lie in perturbations of brain development, which can be manifold, but genetic factors are prominent among these. Genetic studies have pointed to hundreds of causative or susceptibility genes in ASD, making it difficult to find common underlying pathogenic mechanisms. Careful dissection of molecular and cellular mechanisms are needed to define the molecular targets that can translate into therapeutic strategies. On page 1199 of this issue, Bidinosti et al. (1) uncover defects in a molecular machinery of a genetic ASD mouse model. This allowed the authors to design specific chemical interventions that relieve cellular and behavioral autistic-like features. In addition, Yi et al. (2) report a channelopathy in neurons that may predispose to autism. The discoveries raise hope for developing new drugs that help patients with ASD.

Forkhead transcription factors of the FoxO-group are associated with cellular processes like cell cycle progression and DNA-repair. FoxO function is regulated by protein kinase B (PKB) via the phosphatidylinositol 3-kinase/PKB survival pathway. Phosphorylation of serine and threonine residues in specific PKB phosphorylation motifs leads to exclusion of FoxO-proteins from the nucleus, which excludes them from exerting transactivating activity. Members of the FoxO-group have three highly conserved regions containing a PKB phosphorylation motif. This study describes the cloning and characterization of a novel forkhead domain gene from mouse that appeared to be highly related to the FoxO group of transcription factors and was therefore designated FoxO6. The FoxO6 gene was mapped in region D1 on mouse chromosome 4. In humans, FOXO6 is located on chromosomal region 1p34.1. Embryonic expression of FoxO6 is most apparent in the developing brain, and FoxO6 is expressed in a specific temporal and spatial pattern. Therefore it is probably involved in regulation of specific cellular differentiation. In the adult animal FoxO6 expression is maintained in areas of the nucleus accumbens, cingulate cortex, parts of the amygdala, and in the hippocampus. Structure function analysis of FoxO6 compared with its group members shows that the overall homology is high, but surprisingly a highly conserved region containing multiple phosphorylation sites is lacking. In transfection studies, FoxO6 coupled to GFP showed an unexpected high nuclear localization after stimulation with growth factors, in contrast to the predominant cytosolic localization of FoxO1 and FoxO3. We also show that nuclear export of FoxO6 is mediated through the phosphatidylinositol 3-kinase/PKB pathway. Furthermore, we show using a chimeric approach that we can fully restore the ability of FoxO6 to shuttle between nucleus and cytosol. In conclusion, the data presented here gives a new view on regulation of FoxO-function through multiple phosphorylation events and other mechanisms involved in the nuclear exclusion of FoxO-proteins.

Chronic treatment with the opioid antagonist naltrexone induces functional supersensitivity to opioid agonists, which may be explained by receptor up-regulation induced by opioid receptor blockade. In the present study, the levels of opioid receptor subtypes through the brain of mice were determined after chronic naltrexone treatment using quantitative in vitro autoradiography. This is the first complete mapping study in mice for micro-, delta- and kappa-opioid receptors after chronic naltrexone exposure. Treatment with naltrexone clearly induced up-regulation of micro- (mean 80%) and, to a lesser extent, delta-opioid receptors (mean 39%). The up-regulation of micro- and delta-opioid receptors was evident throughout the brain, although there was variation in the percentage change across brain regions. In contrast, consistent up-regulation of kappa-opioid receptors was observed in cortical structures only and was not so marked as for micro- and delta-opioid receptors. In noncortical regions kappa-opioid receptor expression was unchanged. Taken together, the present findings suggest opioid receptor subtype-selective regulation by chronic naltrexone treatment in mice.

Homeobox genes encode transcription factors that play key roles in embryonic development of the nervous system. Dorsal root ganglia are part of the peripheral nervous system that arises from the neural crest. Several homeobox genes are known to play important roles in the development of neural crest and dorsal root ganglia. Some of these remain expressed in the adult dorsal root ganglia (DRG). In order to get more insight into the homeobox gene repertoire in adult rat DRG, we performed RT-PCR with degenerate primers and identified twenty-two homeobox genes. We found homeobox genes that were reported before in embryonic or adult DRG, as well as homeobox genes associated before with neural crest (derivatives). Some of the genes were not reported in relation to neural crest or DRG before. Two homeobox genes displayed sequence differences with their mouse counterparts, possibly being close homologues. The diversity of homeobox genes expressed in adult DRG suggests that gene regulatory events initiated during development remain operative in the mature DRG.

Contactins (Cntns) are a six-member subgroup of the immunoglobulin cell adhesion molecule superfamily (IgCAMs) with pronounced brain expression and function. Recent genetic studies of neuropsychiatric disorders have pinpointed contactin-4 (CNTN4), contactin-5 (CNTN5) and contactin-6 (CNTN6) as candidate genes in neurodevelopmental disorders, particularly in autism spectrum disorders (ASDs), but also in intellectual disability, schizophrenia (SCZ), attention-deficit hyperactivity disorder (ADHD), bipolar disorder (BD), alcohol use disorder (AUD) and anorexia nervosa (AN). This suggests that they have important functions during neurodevelopment. This suggestion is supported by data showing that neurite outgrowth, cell survival and neural circuit formation can be affected by disruption of these genes. Here, we review the current genetic data about their involvement in neuropsychiatric disorders and explore studies on how null mutations affect mouse behavior. Finally, we highlight to role of protein-protein interactions in the potential mechanism of action of Cntn4, -5 and -6 and emphasize that complexes with other membrane proteins may play a role in neuronal developmental functions.

FoxK2 is a forkhead transcription factor expressed ubiquitously in the developing murine central nervous system. Here we investigated the role of FoxK2 in vitro and focused on proliferation and cellular survival. Knockdown of FoxK2 results in a decrease in BrdU incorporation and H3 phosphorylation, suggesting attenuation of proliferation. In the absence of growth factors, FoxK2 knockdown results in a dramatic increase in caspase 3 activity and propidium iodide positive cells, indicative of cell death. Additionally, knockdown of FoxK2 results in an increase in the mRNA of Gadd45α, Gadd45γ, as well as an increase in the phosphorylation of the mTOR dependent kinase p70S6K. Rapamycin treatment completely blocked the increase in p70S6K and synergistically potentiated the decrease in H3 phosphorylation upon FoxK2 knockdown. To gain more insight into the proapoptotic effects upon FoxK2 knockdown we screened for changes in Bcl2 genes. Upon FoxK2 knockdown both Puma and Noxa were significantly upregulated. Both genes were not inhibited by rapamycin treatment, instead rapamycin increased Noxa mRNA. FoxK2 requirement in cellular survival is further emphasized by the fact that resistance to TGFβ-induced cell death was greatly diminished after FoxK2 knockdown. Overall our data suggest FoxK2 is required for proliferation and survival, that mTOR is part of a feedback loop partly compensating for FoxK2 loss, possibly by upregulating Gadd45s, whereas cell death upon FoxK2 loss is induced in a Bcl2 dependent manner via Puma and Noxa.

International audience; Rationale:The genetic heterogeneity of autism spectrum disorders (ASDs) is enormous, and the neurobiology of proteins encoded by genes associated with ASD is very diverse. Revealing the mechanisms on which different neurobiological pathways in ASD pathogenesis converge may lead to the identification of drug targets.; Objective:The main objective is firstly to outline the main molecular networks and neuronal mechanisms in which ASD gene products participate and secondly to answer the question how these converge. Finally, we aim to pinpoint drug targets within these mechanisms.; Method:Literature review of the neurobiological properties of ASD gene products with a special focus on the developmental consequences of genetic defects and the possibility to reverse these by genetic or pharmacological interventions.; Results:The regulation of activity-dependent protein synthesis appears central in the pathogenesis of ASD. Through sequential consequences for axodendritic function, neuronal disabilities arise expressed as behavioral abnormalities and autistic symptoms in ASD patients. Several known ASD gene products have their effect on this central process by affecting protein synthesis intrinsically, e.g., through enhancing the mammalian target of rapamycin (mTOR) signal transduction pathway or through impairing synaptic function in general. These are interrelated processes and can be targeted by compounds from various directions: inhibition of protein synthesis through Lovastatin, mTOR inhibition using rapamycin, or mGluR-related modulation of synaptic activity.; Conclusions:ASD gene products may all feed into a central process of translational control that is important for adequate glutamatergic regulation of dendritic properties. This process can be modulated by available compounds but may also be targeted by yet unexplored routes.

Item does not contain fulltext The establishment of robust and replicable behavioural testing paradigms with translational value for psychiatric diseases is a major step forward in developing and testing etiology-directed treatment for these complex disorders. Based on the existing literature, we have generated an inventory of applied rodent behavioural testing paradigms relevant to autism spectrum disorders (ASD). This inventory focused on previously used paradigms that assess behavioural domains that are affected in ASD, such as social interaction, social communication, repetitive behaviours and behavioural inflexibility, cognition as well as anxiety behaviour. A wide range of behavioural testing paradigms for rodents were identified. However, the level of face and construct validity is highly variable. The predictive validity of these paradigms is unknown, as etiology-directed treatments for ASD are currently not on the market. To optimise these studies, future efforts should address aspects of reproducibility and take into account data about the neurodevelopmental underpinnings and trajectory of ASD. In addition, with the increasing knowledge of processes underlying ASD, such as sensory information processes and synaptic plasticity, phenotyping efforts should include multi-level automated analysis of, for example, representative task-related behavioural and electrophysiological read-outs.