Genoarchitecture of the Early Postmitotic Pretectum and the Role of Wnt Signaling in Shaping Pretectal Neurochemical Anatomy in Zebrafish.

The pretectum has a distinct nuclear arrangement and complex neurochemical anatomy. While previous genoarchitectural studies have described rostrocaudal and dorsoventral progenitor domains and subdomains in different species, the relationship between these early partitions and its later derivatives in the mature anatomy is less understood. The signals and transcription factors that control the establishment of pretectal anatomy are practically unknown. We investigated the possibility that some aspects of the development of pretectal divisions are controlled by Wnt signaling, focusing on the transitional stage between neurogenesis and histogenesis in zebrafish. Using several molecular markers and following the prosomeric model, we identified derivatives from each rostrocaudal pretectal progenitor domain and described the localization of gad1b-positive GABAergic and vglut2.2-positive glutamatergic cell clusters. We also attempted to relate these clusters to pretectal nuclei in the mature brain. Then, we examined the influence of Wnt signaling on the size of neurochemically distinctive pretectal areas, using a chemical inhibitor of the Wnt pathway and the CRISPR/Cas9 approach to knock out genes that encode the Wnt pathway mediators, Lef1 and Tcf7l2. The downregulation of the Wnt pathway led to a decrease in two GABAergic clusters and an expansion of a glutamatergic subregion in the maturing pretectum. This revealed an instructive role of the Wnt signal in the development of the pretectum during neurogenesis. The molecular anatomy presented here improves our understanding of pretectal development during early postmitotic stages and support the hypothesis that Wnt signaling is involved in shaping the neurochemical organization of the pretectum.

Impaired Generation of Transit-Amplifying Progenitors in the Adult Subventricular Zone of Cyclin D2 Knockout Mice.

In the adult brain, new neurons are constitutively derived from postnatal neural stem cells/progenitors located in two neurogenic regions: the subventricular zone (SVZ) of the lateral ventricles (migrating and differentiating into different subtypes of the inhibitory interneurons of the olfactory bulbs), and the subgranular layer of the hippocampal dentate gyrus. Cyclin D2 knockout (cD2-KO) mice exhibit reduced numbers of new hippocampal neurons; however, the proliferation deficiency and the dysregulation of adult neurogenesis in the SVZ required further investigation. In this report, we characterized the differentiation potential of each subpopulation of the SVZ neural precursors in cD2-KO mice. The number of newly generated cells in the SVZs was significantly decreased in cD2-KO mice compared to wild type mice (WT), and was not accompanied by elevated levels of apoptosis. Although the number of B1-type quiescent precursors (B1q) and the overall B1-type activated precursors (B1a) were not affected in the SVZ neurogenic niche, the number of transit-amplifying progenitors (TaPs) was significantly reduced. Additionally, the subpopulations of calbindin D28k and calretinin interneurons were diminished in the olfactory bulbs of cD2-KO mice. Our results suggest that cyclin D2 might be critical for the proliferation of neural precursors and progenitors in the SVZ-the transition of B1a into TaPs and, thereafter, the production of newly generated interneurons in the olfactory bulbs. Untangling regulators that functionally modulate adult neurogenesis provides a basis for the development of regenerative therapies for injuries and neurodegenerative diseases.

Wnt/ß-catenin signaling in brain development and mental disorders: keeping TCF7L2 in mind.

Canonical Wnt signaling, which is transduced by ß-catenin and lymphoid enhancer factor 1/T cell-specific transcription factors (LEF1/TCFs), regulates many aspects of metazoan development and tissue renewal. Although much evidence has associated canonical Wnt/ß-catenin signaling with mood disorders, the mechanistic links are still unknown. Many components of the canonical Wnt pathway are involved in cellular processes that are unrelated to classical canonical Wnt signaling, thus further blurring the picture. The present review critically evaluates the involvement of classical Wnt/ß-catenin signaling in developmental processes that putatively underlie the pathology of mental illnesses. Particular attention is given to the roles of LEF1/TCFs, which have been discussed surprisingly rarely in this context. Highlighting recent discoveries, we propose that alterations in the activity of LEF1/TCFs, and particularly of transcription factor 7-like 2 (TCF7L2), result in defects previously associated with neuropsychiatric disorders, including imbalances in neurogenesis and oligodendrogenesis, the functional disruption of thalamocortical circuitry and dysfunction of the habenula.

TCF7L2 mediates the cellular and behavioral response to chronic lithium treatment in animal models.

The mechanism of lithium's therapeutic action remains obscure, hindering the discovery of safer treatments for bipolar disorder. Lithium can act as an inhibitor of the kinase GSK3α/ß, which in turn negatively regulates ß-catenin, a co-activator of LEF1/TCF transcription factors. However, unclear is whether therapeutic levels of lithium activate ß-catenin in the brain, and whether this activation could have a therapeutic significance. To address this issue we chronically treated mice with lithium. Although the level of non-phospho-ß-catenin increased in all of the brain areas examined, ß-catenin translocated into cellular nuclei only in the thalamus. Similar results were obtained when thalamic and cortical neurons were treated with a therapeutically relevant concentration of lithium in vitro. We tested if TCF7L2, a member of LEF1/TCF family that is highly expressed in the thalamus, facilitated the activation of ß-catenin. Silencing of Tcf7l2 in thalamic neurons prevented ß-catenin from entering the nucleus, even when the cells were treated with lithium. Conversely, when Tcf7l2 was ectopically expressed in cortical neurons, ß-catenin shifted to the nucleus, and lithium augmented this process. Lastly, we silenced tcf7l2 in zebrafish and exposed them to lithium for 3 days, to evaluate whether TCF7L2 is involved in the behavioral response. Lithium decreased the dark-induced activity of control zebrafish, whereas the activity of zebrafish with tcf7l2 knockdown was unaltered. We conclude that therapeutic levels of lithium activate ß-catenin selectively in thalamic neurons. This effect is determined by the presence of TCF7L2, and potentially contributes to the therapeutic response.

Metabolic pathways in the periphery and brain: Contribution to mental disorders?

The association between mental disorders and diabetes has a long history. Recent large-scale, well-controlled epidemiological studies confirmed a link between diabetes and psychiatric illnesses. The scope of this review is to summarize our current understanding of this relationship from a molecular perspective. We first discuss the potential contribution of diabetes-associated metabolic impairments to the etiology of mental conditions. Then, we focus on possible shared molecular risk factors and mechanisms. Simple comorbidity, shared susceptibility loci, and common pathophysiological processes in diabetes and mental illnesses have changed our traditional way of thinking about mental illness. We conclude that schizophrenia and affective disorders are not limited to an imbalance in dopaminergic and serotoninergic neurotransmission in the brain. They are also systemic disorders that can be considered, to some extent, as metabolic disorders.

Molecular anatomy of the thalamic complex and the underlying transcription factors.

Thalamocortical loops have been implicated in the control of higher-order cognitive functions, but advances in our understanding of the molecular underpinnings of neocortical organization have not been accompanied by similar analyses in the thalamus. Using expression-based correlation maps and the manual mapping of mouse and human datasets available in the Allen Brain Atlas, we identified a few individual regions and several sets of molecularly related nuclei that partially overlap with the classic grouping that is based on topographical localization and thalamocortical connections. These new molecular divisions of the adult thalamic complex are defined by the combinatorial expression of Tcf7l2, Lef1, Gbx2, Prox1, Pou4f1, Esrrg, and Six3 transcription factor genes. Further in silico and experimental analyses provided the evidence that TCF7L2 might be a pan-thalamic specifier. These results provide substantial insights into the "molecular logic" that underlies organization of the thalamic complex.

Cognitive flexibility and long-term depression (LTD) are impaired following ß-catenin stabilization in vivo.

The cadherin/ß-catenin adhesion complex is a key mediator of the bidirectional changes in synapse strength which are believed to underlie complex learning and memory. In the present study, we demonstrate that stabilization of ß-catenin in the hippocampus of adult mice results in significant impairments in cognitive flexibility and spatial reversal learning, including impaired extinction during the reversal phase of the Morris water maze and deficits in a delayed nonmatch to place T-maze task. In accordance with these deficits, ß-catenin stabilization was found to abolish long-term depression by stabilizing cadherin at the synaptic membrane and impairing AMPA receptor endocytosis, while leaving basal synaptic transmission and long-term potentiation unaffected. These results demonstrate that the ß-catenin/cadherin adhesion complex plays an important role in learning and memory and that aberrant increases in synaptic adhesion can have deleterious effects on cognitive function.

Physiological role of ß-catenin/TCF signaling in neurons of the adult brain.

Wnt/ß-catenin pathway, the effectors of which are transcription factors of the LEF1/TCF family, is primarily associated with development. Strikingly, however, some of the genes of the pathway are schizophrenia susceptibility genes, and the proteins that are often mutated in neurodegenerative diseases have the ability to regulate ß-catenin levels. If impairment of this pathway indeed leads to these pathologies, then it likely plays a physiological role in the adult brain. This review provides an overview of the current knowledge on this subject. The involvement of ß-catenin and LEF1/TCF factors in adult neurogenesis, synaptic plasticity, and the function of thalamic neurons are discussed. The data are still very preliminary and often based on circumstantial or indirect evidence. Further research might help to understand the etiology of the aforementioned pathologies.

Novel ß-catenin target genes identified in thalamic neurons encode modulators of neuronal excitability.

BACKGROUND: LEF1/TCF transcription factors and their activator ß-catenin are effectors of the canonical Wnt pathway. Although Wnt/ß-catenin signaling has been implicated in neurodegenerative and psychiatric disorders, its possible role in the adult brain remains enigmatic. To address this issue, we sought to identify the genetic program activated by ß-catenin in neurons. We recently showed that ß-catenin accumulates specifically in thalamic neurons where it activates Cacna1g gene expression. In the present study, we combined bioinformatics and experimental approaches to find new ß-catenin targets in the adult thalamus. RESULTS: We first selected the genes with at least two conserved LEF/TCF motifs within the regulatory elements. The resulting list of 428 putative LEF1/TCF targets was significantly enriched in known Wnt targets, validating our approach. Functional annotation of the presumed targets also revealed a group of 41 genes, heretofore not associated with Wnt pathway activity, that encode proteins involved in neuronal signal transmission. Using custom polymerase chain reaction arrays, we profiled the expression of these genes in the rat forebrain. We found that nine of the analyzed genes were highly expressed in the thalamus compared with the cortex and hippocampus. Removal of nuclear ß-catenin from thalamic neurons in vitro by introducing its negative regulator Axin2 reduced the expression of six of the nine genes. Immunoprecipitation of chromatin from the brain tissues confirmed the interaction between ß-catenin and some of the predicted LEF1/TCF motifs. The results of these experiments validated four genes as authentic and direct targets of ß-catenin: Gabra3 for the receptor of GABA neurotransmitter, Calb2 for the Ca(2+)-binding protein calretinin, and the Cacna1g and Kcna6 genes for voltage-gated ion channels. Two other genes from the latter cluster, Cacna2d2 and Kcnh8, appeared to be regulated by ß-catenin, although the binding of ß-catenin to the regulatory sequences of these genes could not be confirmed. CONCLUSIONS: In the thalamus, ß-catenin regulates the expression of a novel group of genes that encode proteins involved in neuronal excitation. This implies that the transcriptional activity of ß-catenin is necessary for the proper excitability of thalamic neurons, may influence activity in the thalamocortical circuit, and may contribute to thalamic pathologies.

WNT protein-independent constitutive nuclear localization of beta-catenin protein and its low degradation rate in thalamic neurons.

Nuclear localization of ß-catenin is a hallmark of canonical Wnt signaling, a pathway that plays a crucial role in brain development and the neurogenesis of the adult brain. We recently showed that ß-catenin accumulates specifically in mature thalamic neurons, where it regulates the expression of the Ca(v)3.1 voltage-gated calcium channel gene. Here, we investigated the mechanisms underlying ß-catenin accumulation in thalamic neurons. We report that a lack of soluble factors produced either by glia or cortical neurons does not impair nuclear ß-catenin accumulation in thalamic neurons. We next found that the number of thalamic neurons with ß-catenin nuclear localization did not change when the Wnt/Dishevelled signaling pathway was inhibited by Dickkopf1 or a dominant negative mutant of Dishevelled3. These results suggest a WNT-independent cell-autonomous mechanism. We found that the protein levels of APC, AXIN1, and GSK3ß, components of the ß-catenin degradation complex, were lower in the thalamus than in the cortex of the adult rat brain. Reduced levels of these proteins were also observed in cultured thalamic neurons compared with cortical cultures. Finally, pulse-chase experiments confirmed that cytoplasmic ß-catenin turnover was slower in thalamic neurons than in cortical neurons. Altogether, our data indicate that the nuclear localization of ß-catenin in thalamic neurons is their cell-intrinsic feature, which was WNT-independent but associated with low levels of proteins involved in ß-catenin labeling for ubiquitination and subsequent degradation.

Differential roles for STIM1 and STIM2 in store-operated calcium entry in rat neurons.

The interaction between Ca(2+) sensors STIM1 and STIM2 and Ca(2+) channel-forming protein ORAI1 is a crucial element of store-operated calcium entry (SOCE) in non-excitable cells. However, the molecular mechanism of SOCE in neurons remains unclear. We addressed this issue by establishing the presence and function of STIM proteins. Real-time polymerase chain reaction from cortical neurons showed that these cells contain significant amounts of Stim1 and Stim2 mRNA. Thapsigargin (TG) treatment increased the amount of both endogenous STIM proteins in neuronal membrane fractions. The number of YFP-STIM1/ORAI1 and YFP-STIM2/ORAI1 complexes was also enhanced by such treatment. The differences observed in the number of STIM1 and STIM2 complexes under SOCE conditions and the differential sensitivity to SOCE inhibitors suggest their distinct roles. Endoplasmic reticulum (ER) store depletion by TG enhanced intracellular Ca(2+) levels in loaded with Fura-2 neurons transfected with YFP-STIM1 and ORAI1, but not with YFP-STIM2 and ORAI1, which correlated well with the number of complexes formed. Moreover, the SOCE inhibitors ML-9 and 2-APB reduced Ca(2+) influx in neurons expressing YFP-STIM1/ORAI1 but produced no effect in cells transfected with YFP-STIM2/ORAI1. Moreover, in neurons transfected with YFP-STIM2/ORAI1, the increase in constitutive calcium entry was greater than with YFP-STIM1/ORAI1. Our data indicate that both STIM proteins are involved in calcium homeostasis in neurons. STIM1 mainly activates SOCE, whereas STIM2 regulates resting Ca(2+) levels in the ER and Ca(2+) leakage with the additional involvement of STIM1.

Morgana/CHP-1 is a novel chaperone able to protect cells from stress.

Morgana/CHP-1 (CHORD containing protein-1) has been recently shown to be necessary for proper cell divisions. However, the presence of the protein in postmitotic tissues such as brain and striated muscle suggests that morgana/CHP-1 has additional cellular functions. Here we show that morgana/CHP-1 behaves like an HSP90 co-chaperone and possesses an independent molecular chaperone activity towards denatured proteins. The expression time profile of morgana/Chp-1 in NIH3T3 cells in response to heat stress is similar to that of Hsp70, a classical effector of Heat Shock Factor-1 mediated stress response. Moreover, overexpression of morgana/CHP-1 in NIH3T3 cells leads to the increased stress resistance of the cells. Interestingly, morgana/Chp-1 upregulation in response to transient global brain ischemia lasts longer in ischemia-resistant regions of the gerbil hippocampus than in vulnerable ones, suggesting the involvement of morgana/CHP-1 in natural protective mechanisms in vivo.

LEF1/beta-catenin complex regulates transcription of the Cav3.1 calcium channel gene (Cacna1g) in thalamic neurons of the adult brain.

beta-Catenin, together with LEF1/TCF transcription factors, activates genes involved in the proliferation and differentiation of neuronal precursor cells. In mature neurons, beta-catenin participates in dendritogenesis and synaptic function as a component of the cadherin cell adhesion complex. However, the transcriptional activity of beta-catenin in these cells remains elusive. In the present study, we found that in the adult mouse brain, beta-catenin and LEF1 accumulate in the nuclei of neurons specifically in the thalamus. The particular electrophysiological properties of thalamic neurons depend on T-type calcium channels. Cav3.1 is the predominant T-type channel subunit in the thalamus, and we hypothesized that the Cacna1g gene encoding Cav3.1 is a target of the LEF1/beta-catenin complex. We demonstrated that the expression of Cacna1g is high in the thalamus and is further increased in thalamic neurons treated in vitro with LiCl or WNT3A, activators of beta-catenin. Luciferase reporter assays confirmed that the Cacna1G promoter is activated by LEF1 and beta-catenin, and footprinting analysis revealed four LEF1 binding sites in the proximal region of this promoter. Chromatin immunoprecipitation demonstrated that the Cacna1g proximal promoter is occupied by beta-catenin in vivo in the thalamus, but not in the hippocampus. Moreover, WNT3A stimulation enhanced T-type current in cultured thalamic neurons. Together, our data indicate that the LEF1/beta-catenin complex regulates transcription of Cacna1g and uncover a novel function for beta-catenin in mature neurons. We propose that beta-catenin contributes to neuronal excitability not only by a local action at the synapse but also by activating gene expression in thalamic neurons.

Biochemical characterization and expression analysis of a novel EF-hand Ca2+ binding protein calmyrin2 (Cib2) in brain indicates its function in NMDA receptor mediated Ca2+ signaling.

Calmyrin2 (CaMy2, Cib2) is a novel EF-hand calcium-binding protein found recently in skeletal muscles. CaMy2 mRNA was also detected in brain, but nothing is known about CaMy2 protein localization and properties in the brain. We report cloning and characterization of CaMy2 in rat brain: its expression pattern, intracellular localization and biochemical features. CaMy2 binds Ca2+ and exhibits Ca2+/conformational switch. Moreover, CaMy2 undergoes N-myristoylation without Ca2+/myristoyl switch, is membrane-associated and localizes in neurons together with Golgi apparatus and dendrite markers. CaMy2 transcript and protein are present mainly in the hippocampus and cortex. In cultured hippocampal neurons, CaMy2 is induced upon neuronal activation. Most prominent increase in CaMy2 protein (7-fold), and mRNA (2-fold) occurs upon stimulation of NMDA receptor (NMDAR). The induction is blocked by translation inhibitors, specific antagonists of NMDAR, the Ca2+-chelator BAPTA, and inhibitors of ERK1/2 and PKC, kinases transmitting NMDAR-linked Ca2+ signal. Our results show that CaMy2 level is controlled by NMDAR and Ca2+ and suggest CaMy2 role in Ca2+ signaling underlying NMDAR activation.

Immunolocalization of STIM1 in the mouse brain.

Capacitative Calcium Entry (CCE) in neurons seems to depend, as in non-excitatory cells, on endoplasmic reticulum calcium sensors STIM1 or STIM2. We show localization of STIM1 in the mouse brain by immunohistochemistry with a specific antibody. STIM1 immunoreactivity has wide, but not uniform, distribution throughout the brain and is observed in neuropil and cells. The most intensive immunoreactivity is observed in Purkinje neurons of cerebellum. High/moderate levels of immunostaining are found in hippocampus, cerebral cortex and in cortico-medial amygdala, low in thalamus and basolateral amygdala. Co-staining with anti-NeuN antibody identify STIM1 immunopositive cells as neurons. Real time PCR demonstrates that Stim2 expression is 7-fold higher than that of Stim1 in hippocampus and 3-fold in other regions. Immunoblotting confirms that levels of STIMs vary in different brain regions. The data show that STIM1 and STIM2 are present in the brain, thus both can be involved in CCE, depending on neuronal type.

Expression of STIM1 in brain and puncta-like co-localization of STIM1 and ORAI1 upon depletion of Ca(2+) store in neurons.

Recent findings indicate that Store Operated Ca(2+) Entry (SOCE) in non-excitable cells is based on the interaction of ER calcium sensor STIM1 with the plasma membrane Ca(2+) channel protein ORAI1. However, despite physiological evidence for functional SOCE in neurons, its mechanism is not known. Using PCR, immunoblotting and immunohistochemical methods we show that STIM1 protein is present in the mouse brain. The protein and mRNA levels of STIM1 are similar in the thalamus, the hippocampus, the cortex and the amygdala and the higher level is observed in the cerebellum. Immunohistochemistry of the cortex and the hippocampus of brain sections shows that STIM1 is present in cell bodies and dendrites of pyramidal neurons. In the cerebellum STIM1 is present in Purkinje and granule cells. The same immunostaining pattern is observed in cultured hippocampal and cortical neurons. Localization of YFP-STIM1 and ORAI1 changes from a dispersed pattern in untreated cortical neurons to puncta-like pattern in cells with a Ca(2+) store depleted by thapsigargin treatment. The YFP-STIM1(D76A) dominant positive mutant, which is active regardless of the Ca(2+) level in ER, concentrates as puncta even without depletion of the neuronal Ca(2+) store. Also, this mutant forces ORAI1 redistribution to form puncta-like staining. We suggest that in neurons, just as in non-excitable cells, the STIM1 and ORAI1 proteins are involved in SOCE.

Dimer composition and promoter context contribute to functional cooperation between AP-1 and NFAT.

The transcription factors activator protein 1 (AP-1) and nuclear factor of activated T-cells (NFAT) cooperate to induce the expression of cytokines during the immune response. While much is known about the signaling pathways and physical interactions between NFAT and AP-1 dimers following lymphocyte activation, few studies have addressed the role of AP-1 composition in modulating NFAT:AP-1-dependent transcription. We examined the function of specific AP-1 complexes using "tethered" AP-1 dimers with defined composition. We found that NFAT can functionally cooperate with all AP-1 dimers tested. Noteworthy, Jun approximately Jun-containing dimers, which are relatively inactive when tested on an AP-1-dependent promoter, are effective co-activators of an NFAT:AP-1-dependent promoter. Interestingly, specific AP-1 dimer combinations behave differently when tested on interleukin 2 (IL2) and interleukin 4 (IL4) gene regulatory regions. Moreover, the requirement for NFAT to activate each of the promoters is different. Our results suggest that higher NFAT levels are necessary to activate the IL4 promoter. Hence changes in AP-1 composition and the level of participating NFAT proteins can differentially influence cytokine gene expression, resulting in biological consequences for the modulation and dynamics of the immune response.

AP-1 dimers regulate transcription of the p14/p19ARF tumor suppressor gene.

Evidence is accumulating about the role of individual AP-1 components in cell proliferation and transformation. Notably, Ras-mediated transformation is characterized by the upregulation of particular AP-1 members, such as c-Jun and Fra-1. The p14/p19ARF tumor suppressor gene is a key link between oncogenic Ras signaling and the p53 pathway. We explored the involvement of AP-1 dimers in the transcriptional regulation of the p14/p19ARF gene. We demonstrate that both the human and mouse ARF promoters are transcriptional targets of selective AP-1 dimers. The ARF promoter is regulated specifically by AP-1 heterodimers containing Fra-1. Overexpression of c-Jun approximately Fra-1 dimers in primary murine fibroblast cells led to the upregulation of the endogenous ARF protein and growth arrest. Conversely, inhibition of c-Jun or Fra-1 protein levels resulted in decreased ARF expression. In addition, we show that AP-1 dimers cooperate with oncogenic Ras in the transcriptional activation of the p14/p19ARF promoter. Thus, AP-1 heterodimers may contribute to the regulation of ARF expression upon oncogenic signaling.