miR-218 Promotes Dopaminergic Differentiation and Controls Neuron Excitability and Neurotransmitter Release through the Regulation of a Synaptic-Related Genes Network.

In the brain, microRNAs (miRNAs) are believed to play a role in orchestrating synaptic plasticity at a higher level by acting as an additional mechanism of translational regulation, alongside the mRNA/polysome system. Despite extensive research, our understanding of the specific contribution of individual miRNA to the function of dopaminergic neurons (DAn) remains limited. By performing a dopaminergic-specific miRNA screening, we have identified miR-218 as a critical regulator of DAn activity in male and female mice. We have found that miR-218 is specifically expressed in mesencephalic DAn and is able to promote dopaminergic differentiation of embryonic stem cells and functional maturation of transdifferentiated induced DA neurons. Midbrain-specific deletion of both genes encoding for miR-218 (referred to as miR-218-1 and mir218-2) affects the expression of a cluster of synaptic-related mRNAs and alters the intrinsic excitability of DAn, as it increases instantaneous frequencies of evoked action potentials, reduces rheobase current, affects the ionic current underlying the action potential after hyperpolarization phase, and reduces dopamine efflux in response to a single electrical stimulus. Our findings provide a comprehensive understanding of the involvement of miR-218 in the dopaminergic system and highlight its role as a modulator of dopaminergic transmission.SIGNIFICANCE STATEMENT In the past decade, several miRNAs have emerged as potential regulators of synapse activity through the modulation of specific gene expression. Among these, we have identified a dopaminergic-specific miRNA, miR-218, which is able to promote dopaminergic differentiation and regulates the translation of an entire cluster of synapse related mRNAs. Deletion of miR-218 has notable effects on dopamine release and alters the intrinsic excitability of dopaminergic neurons, indicating a direct control of dopaminergic activity by miR-218.

MicroRNA-218 instructs proper assembly of hippocampal networks.

The assembly of the mammalian brain is orchestrated by temporally coordinated waves of gene expression. Post-transcriptional regulation by microRNAs (miRNAs) is a key aspect of this program. Indeed, deletion of neuron-enriched miRNAs induces strong developmental phenotypes, and miRNA levels are altered in patients with neurodevelopmental disorders. However, the mechanisms used by miRNAs to instruct brain development remain largely unexplored. Here, we identified miR-218 as a critical regulator of hippocampal assembly. MiR-218 is highly expressed in the hippocampus and enriched in both excitatory principal neurons (PNs) and GABAergic inhibitory interneurons (INs). Early life inhibition of miR-218 results in an adult brain with a predisposition to seizures. Changes in gene expression in the absence of miR-218 suggest that network assembly is impaired. Indeed, we find that miR-218 inhibition results in the disruption of early depolarizing GABAergic signaling, structural defects in dendritic spines, and altered intrinsic membrane excitability. Conditional knockout of Mir218-2 in INs, but not PNs, is sufficient to recapitulate long-term instability. Finally, de-repressing Kif21b and Syt13, two miR-218 targets, phenocopies the effects on early synchronous network activity induced by miR-218 inhibition. Taken together, the data suggest that miR-218 orchestrates formative events in PNs and INs to produce stable networks.

Neuronal dysfunction and gene modulation by non-coding RNA in Parkinson's disease and synucleinopathies.

Over the last few decades, emerging evidence suggests that non-coding RNAs (ncRNAs) including long-non-coding RNA (lncRNA), microRNA (miRNA) and circular-RNA (circRNA) contribute to the molecular events underlying progressive neuronal degeneration, and a plethora of ncRNAs have been identified significantly misregulated in many neurodegenerative diseases, including Parkinson's disease and synucleinopathy. Although a direct link between neuropathology and causative candidates has not been clearly established in many cases, the contribution of ncRNAs to the molecular processes leading to cellular dysfunction observed in neurodegenerative diseases has been addressed, suggesting that they may play a role in the pathophysiology of these diseases. Aim of the present Review is to overview and discuss recent literature focused on the role of RNA-based mechanisms involved in different aspects of neuronal pathology in Parkinson's disease and synucleinopathy models.

Lmx1a-Dependent Activation of miR-204/211 Controls the Timing of Nurr1-Mediated Dopaminergic Differentiation.

The development of midbrain dopaminergic (DA) neurons requires a fine temporal and spatial regulation of a very specific gene expression program. Here, we report that during mouse brain development, the microRNA (miR-) 204/211 is present at a high level in a subset of DA precursors expressing the transcription factor Lmx1a, an early determinant for DA-commitment, but not in more mature neurons expressing Th or Pitx3. By combining different in vitro model systems of DA differentiation, we show that the levels of Lmx1a influence the expression of miR-204/211. Using published transcriptomic data, we found a significant enrichment of miR-204/211 target genes in midbrain dopaminergic neurons where Lmx1a was selectively deleted at embryonic stages. We further demonstrated that miR-204/211 controls the timing of the DA differentiation by directly downregulating the expression of Nurr1, a late DA differentiation master gene. Thus, our data indicate the Lmx1a-miR-204/211-Nurr1 axis as a key component in the cascade of events that ultimately lead to mature midbrain dopaminergic neurons differentiation and point to miR-204/211 as the molecular switch regulating the timing of Nurr1 expression.

Nucleolar localization of the ErbB3 receptor as a new target in glioblastoma.

BACKGROUND: The nucleolus is a subnuclear, non-membrane bound domain that is the hub of ribosome biogenesis and a critical regulator of cell homeostasis. Rapid growth and division of cells in tumors are correlated with intensive nucleolar metabolism as a response to oncogenic factors overexpression. Several members of the Epidermal Growth Factor Receptor (EGFR) family, have been identified in the nucleus and nucleolus of many cancer cells, but their function in these compartments remains unexplored. RESULTS: We focused our research on the nucleolar function that a specific member of EGFR family, the ErbB3 receptor, plays in glioblastoma, a tumor without effective therapies. Here, Neuregulin 1 mediated proliferative stimuli, promotes ErbB3 relocalization from the nucleolus to the cytoplasm and increases pre-rRNA synthesis. Instead ErbB3 silencing or nucleolar stress reduce cell proliferation and affect cell cycle progression. CONCLUSIONS: These data point to the existence of an ErbB3-mediated non canonical pathway that glioblastoma cells use to control ribosomes synthesis and cell proliferation. These results highlight the potential role for the nucleolar ErbB3 receptor, as a new target in glioblastoma.

Altered heparan sulfate metabolism during development triggers dopamine-dependent autistic-behaviours in models of lysosomal storage disorders.

Lysosomal storage disorders characterized by altered metabolism of heparan sulfate, including Mucopolysaccharidosis (MPS) III and MPS-II, exhibit lysosomal dysfunctions leading to neurodegeneration and dementia in children. In lysosomal storage disorders, dementia is preceded by severe and therapy-resistant autistic-like symptoms of unknown cause. Using mouse and cellular models of MPS-IIIA, we discovered that autistic-like behaviours are due to increased proliferation of mesencephalic dopamine neurons originating during embryogenesis, which is not due to lysosomal dysfunction, but to altered HS function. Hyperdopaminergia and autistic-like behaviours are corrected by the dopamine D1-like receptor antagonist SCH-23390, providing a potential alternative strategy to the D2-like antagonist haloperidol that has only minimal therapeutic effects in MPS-IIIA. These findings identify embryonic dopaminergic neurodevelopmental defects due to altered function of HS leading to autistic-like behaviours in MPS-II and MPS-IIIA and support evidence showing that altered HS-related gene function is causative of autism.

Molecular Regulation in Dopaminergic Neuron Development. Cues to Unveil Molecular Pathogenesis and Pharmacological Targets of Neurodegeneration.

The relatively few dopaminergic neurons in the mammalian brain are mostly located in the midbrain and regulate many important neural functions, including motor integration, cognition, emotive behaviors and reward. Therefore, alteration of their function or degeneration leads to severe neurological and neuropsychiatric diseases. Unraveling the mechanisms of midbrain dopaminergic (mDA) phenotype induction and maturation and elucidating the role of the gene network involved in the development and maintenance of these neurons is of pivotal importance to rescue or substitute these cells in order to restore dopaminergic functions. Recently, in addition to morphogens and transcription factors, microRNAs have been identified as critical players to confer mDA identity. The elucidation of the gene network involved in mDA neuron development and function will be crucial to identify early changes of mDA neurons that occur in pre-symptomatic pathological conditions, such as Parkinson's disease. In addition, it can help to identify targets for new therapies and for cell reprogramming into mDA neurons. In this essay, we review the cascade of transcriptional and posttranscriptional regulation that confers mDA identity and regulates their functions. Additionally, we highlight certain mechanisms that offer important clues to unveil molecular pathogenesis of mDA neuron dysfunction and potential pharmacological targets for the treatment of mDA neuron dysfunction.

Correction: Direct Regulation of Pitx3 Expression by Nurr1 in Culture and in Developing Mouse Midbrain.

[This corrects the article DOI: 10.1371/journal.pone.0030661.].

miR-218 Inhibits Mitochondrial Clearance by Targeting PRKN E3 Ubiquitin Ligase.

The selective elimination of dysfunctional mitochondria through mitophagy is crucial for preserving mitochondrial quality and cellular homeostasis. The most described mitophagy pathway is regulated by a positive ubiquitylation feedback loop in which the PINK1 (PTEN induced kinase 1) kinase phosphorylates both ubiquitin and the E3 ubiquitin ligase PRKN (Parkin RBR E3 ubiquitin ligase), also known as PARKIN. This event recruits PRKN to the mitochondria, thus amplifying ubiquitylation signal. Here we report that miR-218 targets PRKN and negatively regulates PINK1/PRKN-mediated mitophagy. Overexpression of miR-218 reduces PRKN mRNA levels, thus also reducing protein content and deregulating the E3 ubiquitin ligase action. In fact, following miR-218 overexpression, mitochondria result less ubiquitylated and the autophagy machinery fails to proceed with correct mitochondrial clearance. Since mitophagy defects are associated with various human diseases, these results qualify miR-218 as a promising therapeutic target for human diseases.

miR-34b/c Regulates Wnt1 and Enhances Mesencephalic Dopaminergic Neuron Differentiation.

The differentiation of dopaminergic neurons requires concerted action of morphogens and transcription factors acting in a precise and well-defined time window. Very little is known about the potential role of microRNA in these events. By performing a microRNA-mRNA paired microarray screening, we identified miR-34b/c among the most upregulated microRNAs during dopaminergic differentiation. Interestingly, miR-34b/c modulates Wnt1 expression, promotes cell cycle exit, and induces dopaminergic differentiation. When combined with transcription factors ASCL1 and NURR1, miR-34b/c doubled the yield of transdifferentiated fibroblasts into dopaminergic neurons. Induced dopaminergic (iDA) cells synthesize dopamine and show spontaneous electrical activity, reversibly blocked by tetrodotoxin, consistent with the electrophysiological properties featured by brain dopaminergic neurons. Our findings point to a role for miR-34b/c in neuronal commitment and highlight the potential of exploiting its synergy with key transcription factors in enhancing in vitro generation of dopaminergic neurons.

A meta-analytic approach to genes that are associated with impaired and elevated spatial memory performance.

Spatial memory deficits are a common hallmark of psychiatric conditions, possibly due to a genetic predisposition. Thus, unravelling the relationship between genes and memory might suggest novel therapeutic targets and pathogenetic pathways. Genetic deletions are known to lead to memory deficits (post-deletion "forgetfulness" genes, PDF), or, in few instances to improve spatial memory (post-deletion "hypermnesic" genes, PDH). To assess this topic, we performed a meta-analytic approach on memory behavior in knock-out mice. We screened 300 studies from PubMed and retrieved 87 genes tested for possible effects on spatial memory. This database was crossed with the Allen Brain Atlas (brain distribution) and the Enrichr (gene function) databases. The results show that PDF genes have higher expression level in several ventral brain structures, particularly the encephalic trunk and in the hypothalamus. Moreover, part of these genes are implicated in synaptic functions. Conversely, the PDH genes are associated to G-protein coupled receptors downstream signalling. Some candidate drugs were also found to interfere with some of the PDH genes, further suggesting that this approach might help in identifying drugs to improve memory performance in psychiatric conditions.

Information content of dendritic spines after motor learning.

Dendritic spines, small protrusions emerging from the dendrites of most excitatory synapses in the mammalian brain, are highly dynamic structures and their shape and number is continuously modulated by memory formation and other adaptive changes of the brain. In this study, using a behavioral paradigm of motor learning, we applied the non-linear analysis of dendritic spines to study spine complexity along dendrites of cortical and subcortical neural systems, such as the basal ganglia, that sustain important motor learning processes. We show that, after learning, the spine organization has greater complexity, as indexed by the maximum Lyapunov exponent (LyE). The positive value of the exponent demonstrates that the system is chaotic, while recurrence plots show that the system is not simply composed by random noise, but displays quasi-periodic behavior. The increase in the maximum LyE and in the system entropy after learning was confirmed by the modification of the reconstructed trajectories in phase-space. Our results suggest that the remodeling of spines, as a result of a chaotic and non-random dynamical process along dendrites, may be a general feature associated with the structural plasticity underlying processes such as long-term memory maintenance. Furthermore, this work indicates that the non-linear method is a very useful tool to allow the detection of subtle stimulus-induced changes in dendritic spine dynamics, giving a key contribution to the study of the relationship between structure and function of spines.

Motor learning and metaplasticity in striatal neurons: relevance for Parkinson's disease.

Nigro-striatal dopamine transmission is central to a wide range of neuronal functions, including skill learning, which is disrupted in several pathologies such as Parkinson's disease. The synaptic plasticity mechanisms, by which initial motor learning is stored for long time periods in striatal neurons, to then be gradually optimized upon subsequent training, remain unexplored. Addressing this issue is crucial to identify the synaptic and molecular mechanisms involved in striatal-dependent learning impairment in Parkinson's disease. In this study, we took advantage of interindividual differences between outbred rodents in reaching plateau performance in the rotarod incremental motor learning protocol, to study striatal synaptic plasticity ex vivo. We then assessed how this process is modulated by dopamine receptors and the dopamine active transporter, and whether it is impaired by overexpression of human α-synuclein in the mesencephalon; the latter is a progressive animal model of Parkinson's disease. We found that the initial acquisition of motor learning induced a dopamine active transporter and D1 receptors mediated long-term potentiation, under a protocol of long-term depression in striatal medium spiny neurons. This effect disappeared in animals reaching performance plateau. Overexpression of human α-synuclein reduced striatal dopamine active transporter levels, impaired motor learning, and prevented the learning-induced long-term potentiation, before the appearance of dopamine neuronal loss. Our findings provide evidence of a reorganization of cellular plasticity within the dorsolateral striatum that is mediated by dopamine receptors and dopamine active transporter during the acquisition of a skill. This newly identified mechanism of cellular memory is a form of metaplasticity that is disrupted in the early stage of synucleinopathies, such as Parkinson's disease, and that might be relevant for other striatal pathologies, such as drug abuse.

Glycosphingolipid metabolic reprogramming drives neural differentiation.

Neural development is accomplished by differentiation events leading to metabolic reprogramming. Glycosphingolipid metabolism is reprogrammed during neural development with a switch from globo- to ganglio-series glycosphingolipid production. Failure to execute this glycosphingolipid switch leads to neurodevelopmental disorders in humans, indicating that glycosphingolipids are key players in this process. Nevertheless, both the molecular mechanisms that control the glycosphingolipid switch and its function in neurodevelopment are poorly understood. Here, we describe a self-contained circuit that controls glycosphingolipid reprogramming and neural differentiation. We find that globo-series glycosphingolipids repress the epigenetic regulator of neuronal gene expression AUTS2. AUTS2 in turn binds and activates the promoter of the first and rate-limiting ganglioside-producing enzyme GM3 synthase, thus fostering the synthesis of gangliosides. By this mechanism, the globo-AUTS2 axis controls glycosphingolipid reprogramming and neural gene expression during neural differentiation, which involves this circuit in neurodevelopment and its defects in neuropathology.

Serotonin 5-HT7 receptor increases the density of dendritic spines and facilitates synaptogenesis in forebrain neurons.

Precise control of dendritic spine density and synapse formation is critical for normal and pathological brain functions. Therefore, signaling pathways influencing dendrite outgrowth and remodeling remain a subject of extensive investigations. Here, we report that prolonged activation of the serotonin 5-HT7 receptor (5-HT7R) with selective agonist LP-211 promotes formation of dendritic spines and facilitates synaptogenesis in postnatal cortical and striatal neurons. Critical role of 5-HT7R in neuronal morphogenesis was confirmed by analysis of neurons isolated from 5-HT7R-deficient mice and by pharmacological inactivation of the receptor. Acute activation of 5-HT7R results in pronounced neurite elongation in postnatal striatal and cortical neurons, thus extending previous data on the morphogenic role of 5-HT7R in embryonic and hippocampal neurons. We also observed decreased number of spines in neurons with either genetically (i.e. 5-HT7R-knock-out) or pharmacologically (i.e. antagonist treatment) blocked 5-HT7R, suggesting that constitutive 5-HT7R activity is critically involved in the spinogenesis. Moreover, cyclin-dependent kinase 5 and small GTPase Cdc42 were identified as important downstream effectors mediating morphogenic effects of 5-HT7R in neurons. Altogether, our data suggest that the 5-HT7R-mediated structural reorganization during the postnatal development might have a crucial role for the development and plasticity of forebrain areas such as cortex and striatum, and thereby can be implicated in regulation of the higher cognitive functions. Read the Editorial Highlight for this article on page 644.

Production of Small Noncoding RNAs from the flamenco Locus Is Regulated by the gypsy Retrotransposon of Drosophila melanogaster.

Protective mechanisms based on RNA silencing directed against the propagation of transposable elements are highly conserved in eukaryotes. The control of transposable elements is mediated by small noncoding RNAs, which derive from transposon-rich heterochromatic regions that function as small RNA-generating loci. These clusters are transcribed and the precursor transcripts are processed to generate Piwi-interacting RNAs (piRNAs) and endogenous small interfering RNAs (endo-siRNAs), which silence transposable elements in gonads and somatic tissues. The flamenco locus is a Drosophila melanogaster small RNA cluster that controls gypsy and other transposable elements, and has played an important role in understanding how small noncoding RNAs repress transposable elements. In this study, we describe a cosuppression mechanism triggered by new euchromatic gypsy insertions in genetic backgrounds carrying flamenco alleles defective in gypsy suppression. We found that the silencing of gypsy is accompanied by the silencing of other transposons regulated by flamenco, and of specific flamenco sequences from which small RNAs against gypsy originate. This cosuppression mechanism seems to depend on a post-transcriptional regulation that involves both endo-siRNA and piRNA pathways and is associated with the occurrence of developmental defects. In conclusion, we propose that new gypsy euchromatic insertions trigger a post-transcriptional silencing of gypsy sense and antisense sequences, which modifies the flamenco activity. This cosuppression mechanism interferes with some developmental processes, presumably by influencing the expression of specific genes.

The transcription factor EGR1 localizes to the nucleolus and is linked to suppression of ribosomal precursor synthesis.

EGR1 is an immediate early gene with a wide range of activities as transcription factor, spanning from regulation of cell growth to differentiation. Numerous studies show that EGR1 either promotes the proliferation of stimulated cells or suppresses the tumorigenic growth of transformed cells. Upon interaction with ARF, EGR1 is sumoylated and acquires the ability to bind to specific targets such as PTEN and in turn to regulate cell growth. ARF is mainly localized to the periphery of nucleolus where is able to negatively regulate ribosome biogenesis. Since EGR1 colocalizes with ARF under IGF-1 stimulation we asked the question of whether EGR1 also relocate to the nucleolus to interact with ARF. Here we show that EGR1 colocalizes with nucleolar markers such as fibrillarin and B23 in the presence of ARF. Western analysis of nucleolar extracts from HeLa cells was used to confirm the presence of EGR1 in the nucleolus mainly as the 100 kDa sumoylated form. We also show that the level of the ribosomal RNA precursor 47S is inversely correlated to the level of EGR1 transcripts. The EGR1 iseffective to regulate the synthesis of the 47S rRNA precursor. Then we demonstrated that EGR1 binds to the Upstream Binding Factor (UBF) leading us to hypothesize that the regulating activity of EGR1 is mediated by its interaction within the transcriptional complex of RNA polymerase I. These results confirm the presence of EGR1 in the nucleolus and point to a role for EGR1 in the control of nucleolar metabolism.

What dictates the accumulation of desmosterol in the developing brain?

The brain is the most cholesterol-enriched tissue in the body. During brain development, desmosterol, an immediate precursor of cholesterol, transiently accumulates up to 30% of total brain sterols. This massive desmosterol deposition appears to be present in all mammalian species reported so far, including humans, but how it is achieved is not well understood. Here, we propose that desmosterol accumulation in the developing brain may be primarily caused by post-transcriptional repression of 3ß-hydroxysterol 24-reductase (DHCR24) by progesterone. Furthermore, distinct properties of desmosterol may serve to increase the membrane active pool of sterols in the brain: desmosterol cannot be hydroxylated to generate 24S-hydroxycholesterol, a brain derived secretory sterol, desmosterol has a reduced propensity to be esterified as compared to cholesterol, and desmosterol may activate LXR to stimulate astrocyte sterol secretion. This regulated accumulation of desmosterol by progesterone-induced suppression of DHCR24 may facilitate the rapid enrichment and distribution of membrane sterols in the developing brain.

Adult neural stem cells: an endogenous tool to repair brain injury?

Research on stem cells has developed as one of the most promising areas of neurobiology. In the beginning of the 1990s, neurogenesis in the adult brain was indisputably accepted, eliciting great research efforts. Neural stem cells in the adult mammalian brain are located in the 'neurogenic' areas of the subventricular and subgranular zones. Nevertheless, many reports indicate that they subsist in other regions of the adult brain. Adult neural stem cells have arisen considerable interest as these studies can be useful to develop new methods to replace damaged neurons and treat severe neurological diseases such as neurodegeneration, stroke or spinal cord lesions. In particular, a promising field is aimed at stimulating or trigger a self-repair system in the diseased brain driven by its own stem cell population. Here, we will revise the latest findings on the characterization of active and quiescent adult neural stem cells in the main regions of neurogenesis and the factors necessary to maintain their active and resting states, stimulate migration and homing in diseased areas, hoping to outline the emerging knowledge for the promotion of regeneration in the brain based on endogenous stem cells.

Direct regulation of Pitx3 expression by Nurr1 in culture and in developing mouse midbrain.

Due to their correlation with major human neurological diseases, dopaminergic neurons are some of the most studied neuronal subtypes. Mesencephalic dopaminergic (mDA) differentiation requires the activation of a cascade of transcription factors, among which play a crucial role the nuclear receptor Nurr1 and the paired-like homeodomain 3, Pitx3. During development the expression of Nurr1 precedes that of Pitx3 and those of typical dopaminergic markers such as tyrosine hydroxylase (TH) and dopamine Transporter (DAT) that are directly regulated by Nurr1. Interestingly we have previously demonstrated that Nurr1 RNA silencing reduced Pitx3 transcripts, leading to the hypothesis that Nurr1 may control Pitx3 expression.Here we show that Nurr1 overexpression up-regulates that of Pitx3 in a dose-dependent manner by binding to a non-canonical NBRE consensus sequence, located at the 5' site of the gene. Interestingly, this sequence shows the same effect as the canonical one in promoting gene translation, and its deletion abolishes the ability of Nurr1 to sustain reporter gene expression. Moreover, we show that there is a direct interaction between Nurr1 and the Pitx3 gene promoter in dopaminergic cell cultures and midbrain embryonic tissue. Altogether, our results suggest that the regulation of Pitx3 by Nurr1 may be an essential event controlling the development and function of mDA neurons.