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.

Selymatra: A web application for protein-profiling analysis of mass spectra.

Surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry is a variant of the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. It is used in many cases especially for the analysis of protein profiling and for preliminary screening of biomarkers in complex samples. Unfortunately, these analyses are time consuming and protein identification is generally strictly limited. SELDI-TOF analysis of mass spectra (SELYMATRA) is a web application (WA) developed to reduce these limitations by (i) automating the identification processes and (ii) introducing the possibility to predict proteins in complex mixtures from cells and tissues. The WA architectural pattern is the model-view-controller, commonly used in software development. The WA compares the mass value between two mass spectra (sample vs. control) to extract differences, and, according to the set parameters, it queries a local database to predict most likely proteins based on their masses and different expression amplification. The WA was validated in a cellular model overexpressing a tagged NURR1 receptor, being able to recognize the tagged protein in the profiling of transformed cells. A help page, including a description of parameters for WA use, is available on the website.

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.

Modulation of neuronal morphology by antipsychotic drug: Involvement of serotonin receptor 7.

Antipsychotic drugs (APDs) are the primary pharmacological treatment for schizophrenia, a complex disorder characterized by altered neuronal connectivity. Atypical or second-generation antipsychotics, such as Risperidone (RSP) and Clozapine (CZP) predominantly block dopaminergic D2 and serotonin receptor 2A (5-HT2A) neurotransmission. Both compounds also exhibit affinity for the 5-HT7R, with RSP acting as an antagonist and CZP as an inverse agonist. Our study aimed to determine whether RSP and CZP can influence neuronal morphology through a 5-HT7R-mediated mechanism. Here, we demonstrated that CZP promotes neurite outgrowth of early postnatal cortical neurons, and the 5-HT7R mediates its effect. Conversely, RSP leads to a reduction of neurite length of early postnatal cortical neurons, in a 5-HT7R-independent way. Furthermore, we found that the effects of CZP, mediated by 5-HT7R activation, require the participation of ERK and Cdk5 kinase pathways. At the same time, the modulation of neurite length by RSP does not involve these pathways. In conclusion, our findings provide valuable insights into the morphological changes induced by these two APDs in neurons and elucidate some of the associated molecular pathways. Investigating the 5-HT7R-dependent signaling pathways underlying the neuronal morphogenic effects of APDs may contribute to the identification of novel targets for the treatment of schizophrenia.

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.

Combined DiI and Antibody Labeling Reveals Complex Dysgenesis of Hippocampal Dendritic Spines in a Mouse Model of Fragile X Syndrome.

Structural, functional, and molecular alterations in excitatory spines are a common hallmark of many neurodevelopmental disorders including intellectual disability and autism. Here, we describe an optimized methodology, based on combined use of DiI and immunofluorescence, for rapid and sensitive characterization of the structure and composition of spines in native brain tissue. We successfully demonstrate the applicability of this approach by examining the properties of hippocampal spines in juvenile Fmr1 KO mice, a mouse model of Fragile X Syndrome. We find that mutant mice display pervasive dysgenesis of spines evidenced by an overabundance of both abnormally elongated thin spines and cup-shaped spines, in combination with reduced density of mushroom spines. We further find that mushroom spines expressing the actin-binding protein Synaptopodin-a marker for spine apparatus-are more prevalent in mutant mice. Previous work identified spines with Synaptopodin/spine apparatus as the locus of mGluR-LTD, which is abnormally elevated in Fmr1 KO mice. Altogether, our data suggest this enhancement may be linked to the preponderance of this subset of spines in the mutant. Overall, these findings demonstrate the sensitivity and versatility of the optimized methodology by uncovering a novel facet of spine dysgenesis in Fmr1 KO mice.

Music affects functional brain connectivity and is effective in the treatment of neurological disorders.

In a million years, under the pressure of natural selection, hominins have acquired the abilities for vocal learning, music, and language. Music is a relevant human activity, highly effective in enhancing sociality, is a universal experience common to all known human cultures, although it varies in rhythmic and melodic complexity. It has been part of human life since the beginning of our history, or almost, and it strengthens the mother-baby relation even within the mother's womb. Music engages multiple cognitive functions, and promotes attention, concentration, imagination, creativity, elicits memories and emotions, and stimulates imagination, and harmony of movement. It changes the chemistry of the brain, by inducing the release of neurotransmitters and hormones (dopamine, serotonin, and oxytocin) and activates the reward and prosocial systems. In addition, music is also used to develop new therapies necessary to alleviate severe illness, especially neurological disorders, and brain injuries.

SINEUPs: a novel toolbox for RNA therapeutics.

RNA molecules have emerged as a new class of promising therapeutics to expand the range of druggable targets in the genome. In addition to 'canonical' protein-coding mRNAs, the emerging richness of sense and antisense long non-coding RNAs (lncRNAs) provides a new reservoir of molecular tools for RNA-based drugs. LncRNAs are composed of modular structural domains with specific activities involving the recruitment of protein cofactors or directly interacting with nucleic acids. A single therapeutic RNA transcript can then be assembled combining domains with defined secondary structures and functions, and antisense sequences specific for the RNA/DNA target of interest. As the first representative molecules of this new pharmacology, we have identified SINEUPs, a new functional class of natural antisense lncRNAs that increase the translation of partially overlapping mRNAs. Their activity is based on the combination of two domains: an embedded mouse inverted SINEB2 element that enhances mRNA translation (effector domain) and an overlapping antisense region that provides specificity for the target sense transcript (binding domain). By genetic engineering, synthetic SINEUPs can potentially target any mRNA of interest increasing translation and therefore the endogenous level of the encoded protein. In this review, we describe the state-of-the-art knowledge of SINEUPs and discuss recent publications showing their potential application in diseases where a physiological increase of endogenous protein expression can be therapeutic.

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.

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.].

Transdifferentiation of Mouse Embryonic Fibroblasts into Dopaminergic Neurons Reactivates LINE-1 Repetitive Elements.

In mammals, LINE-1 (L1) retrotransposons constitute between 15% and 20% of the genome. Although only a few copies have retained the ability to retrotranspose, evidence in brain and differentiating pluripotent cells indicates that L1 retrotransposition occurs and creates mosaics in normal somatic tissues. The function of de novo insertions remains to be understood. The transdifferentiation of mouse embryonic fibroblasts to dopaminergic neuronal fate provides a suitable model for studying L1 dynamics in a defined genomic and unaltered epigenomic background. We found that L1 elements are specifically re-expressed and mobilized during the initial stages of reprogramming and that their insertions into specific acceptor loci coincides with higher chromatin accessibility and creation of new transcribed units. Those events accompany the maturation of neuronal committed cells. We conclude that L1 retrotransposition is a non-random process correlating with chromatin opening and lncRNA production that accompanies direct somatic cell reprogramming.

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.

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.