Aron Kos

MicroRNA networks direct neuronal development and plasticity

MicroRNAs (miRNAs) constitute a class of small, non-coding RNAs that act as post-transcriptional regulators of gene expression. In neurons, the functions of individual miRNAs are just beginning to emerge, and recent studies have elucidated roles for neural miRNAs at various stages of neuronal development and maturation, including neurite outgrowth, dendritogenesis, and spine formation. Notably, miRNAs regulate mRNA translation locally in the axosomal and synaptodendritic compartments, and thereby contribute to the dynamic spatial organization of axonal and dendritic structures and their function. Given the critical role for miRNAs in regulating early brain development and in mediating synaptic plasticity later in life, it is tempting to speculate that the pathology of neurological disorders is affected by altered expression or functioning of miRNAs. Here we provide an overview of recently identified mechanisms of neuronal development and plasticity involving miRNAs, and the consequences of miRNA dysregulation.

Chromosome 1p21.3 microdeletions comprising DPYD and MIR137 are associated with intellectual disability

Background MicroRNAs (miRNAs) are non-coding gene transcripts involved in post-transcriptional regulation of genes. Recent studies identified miRNAs as important regulators of learning and memory in model organisms. So far, no mutations in specific miRNA genes have been associated with impaired cognitive functions. Methods and results In three sibs and two unrelated patients with intellectual disability (ID), overlapping 1p21.3 deletions were detected by genome-wide array analysis. The shortest region of overlap included dihydropyrimidine dehydrogenase (DPYD) and microRNA 137 (MIR137). DPYD is involved in autosomal recessive dihydropyrimidine dehydrogenase deficiency. Hemizygous DPYD deletions were previously suggested to contribute to a phenotype with autism spectrum disorder and speech delay. Interestingly, the mature microRNA transcript microRNA-137 (miR-137) was recently shown to be involved in modulating neurogenesis in adult murine neuronal stem cells. Therefore, this study investigated the possible involvement of MIR137 in the 1p21.3-deletion phenotype. The patients displayed a significantly decreased expression of both precursor and mature miR-137 levels, as well as significantly increased expression of the validated downstream targets microphthalmia-associated transcription factor (MITF) and Enhancer of Zeste, Drosophila, Homologue 2 (EZH2), and the newly identified target Kruppel-like factor 4 (KLF4). The study also demonstrated significant enrichment of miR-137 at the synapses of cortical and hippocampal neurons, suggesting a role of miR-137 in regulating local synaptic protein synthesis machinery. Conclusions This study showed that dosage effects of MIR137 are associated with 1p21.3 microdeletions and may therefore contribute to the ID phenotype in patients with deletions harbouring this miRNA. A local effect at the synapse might be responsible.

A potential regulatory role for intronic microRNA-338-3p for its host gene encoding apoptosis-associated tyrosine kinase.

MicroRNAs (miRNAs) are important gene regulators that are abundantly expressed in both the developing and adult mammalian brain. These non-coding gene transcripts are involved in post-transcriptional regulatory processes by binding to specific target mRNAs. Approximately one third of known miRNA genes are located within intronic regions of protein coding and non-coding regions, and previous studies have suggested a role for intronic miRNAs as negative feedback regulators of their host genes. In the present study, we monitored the dynamic gene expression changes of the intronic miR-338-3p and miR-338-5p and their host gene Apoptosis-associated Tyrosine Kinase (AATK) during the maturation of rat hippocampal neurons. This revealed an uncorrelated expression pattern of mature miR-338 strands with their host gene. Sequence analysis of the 3′ untranslated region (UTR) of rat AATK mRNA revealed the presence of two putative binding sites for miR-338-3p. Thus, miR-338-3p may have the capacity to modulate AATK mRNA levels in neurons. Transfection of miR-338-3p mimics into rat B35 neuroblastoma cells resulted in a significant decrease of AATK mRNA levels, while the transfection of synthetic miR-338-5p mimics did not alter AATK levels. Our results point to a possible molecular mechanism by which miR-338-3p participates in the regulation of its host gene by modulating the levels of AATK mRNA, a kinase which plays a role during differentiation, apoptosis and possibly in neuronal degeneration.

Microrna-137 controls ampa-receptor-mediated transmission and mglur-dependent ltd

Mutations affecting the levels of microRNA miR-137 are associated with intellectual disability and schizophrenia. However, the pathophysiological role of miR-137 remains poorly understood. Here, we describe a highly conserved miR-137-binding site within the mRNA encoding the GluA1 subunit of AMPA-type glutamate receptors (AMPARs) and confirm that GluA1 is a direct target of miR-137. Postsynaptic downregulation of miR-137 at the CA3-CA1 hippocampal synapse selectively enhances AMPAR-mediated synaptic transmission and converts silent synapses to active synapses. Conversely, miR-137 overexpression selectively reduces AMPAR-mediated synaptic transmission and silences active synapses. In addition, we find that miR-137 is transiently upregulated in response to metabotropic glutamate receptor 5 (mGluR5), but not mGluR1 activation. Consequently, acute interference with miR-137 function impedes mGluR-LTD expression. Our findings suggest that miR-137 is a key factor in the control of synaptic efficacy and mGluR-dependent synaptic plasticity, supporting the notion that glutamatergic dysfunction contributes to the pathogenesis of miR-137-linked cognitive impairments.

Breaking limitations of complex culture media: Functional non-viral miRNA delivery into pharmaceutical production cell lines

MicroRNAs (miRNAs) are promising targets for cell engineering through modulation of crucial cellular pathways. An effective introduction of miRNAs into the cell is a prerequisite to reliably study microRNA function. Previously, non-viral delivery of nucleic acids has been demonstrated to be cell type as well as culture medium dependent. Due to their importance for biopharmaceutical research and manufacturing, Chinese hamster ovary (CHO) and Cevecs Amniocyte Production (CAP) cells were used as host cell lines to investigate transfection reagents with respect to successful delivery of small non-coding RNAs (ncRNAs) and their ability to allow for biological activity of miRNAs and small interfering RNAs (siRNAs) within the cell. In the present study, we screened numerous transfection reagents for their suitability to successfully deliver miRNA mimics into CHO DG44 and CAP cells. Our investigation revealed that the determination of transfection efficiency for a given transfection reagent alone is not sufficient to draw conclusions about its ability to maintain the functionality of the miRNA. We could show that independent from high transfection rates observed for several reagents only one was suitable for efficient introduction of functional miRNA mimics into cells cultured in complex protein production media. We provide evidence for the functionality of transferred ncRNAs by demonstrating siRNA-mediated changes in protein levels and cellular phenotype as well as decreased twinfilin-1 (twf-1) transcript levels by its upstream miR-1 regulator. Furthermore, the process could be shown to be scalable which has important implications for biotechnological applications.

Elevated microRNA-181c and microRNA-30d levels in the enlarged amygdala of the valproic acid rat model of autism ☆

Autism spectrum disorders are severe neurodevelopmental disorders, marked by impairments in reciprocal social interaction, delays in early language and communication, and the presence of restrictive, repetitive and stereotyped behaviors. Accumulating evidence suggests that dysfunction of the amygdala may be partially responsible for the impairment of social behavior that is a hallmark feature of ASD. Our studies suggest that a valproic acid (VPA) rat model of ASD exhibits an enlargement of the amygdala as compared to controls rats, similar to that observed in adolescent ASD individuals. Since recent research suggests that altered neuronal development and morphology, as seen in ASD, may result from a common post-transcriptional process that is under tight regulation by microRNAs (miRs), we examined genome-wide transcriptomics expression in the amygdala of rats prenatally exposed to VPA, and detected elevated miR-181c and miR-30d expression levels as well as dysregulated expression of their cognate mRNA targets encoding proteins involved in neuronal system development. Furthermore, selective suppression of miR-181c function attenuates neurite outgrowth and branching, and results in reduced synaptic density in primary amygdalar neurons in vitro. Collectively, these results implicate the small non-coding miR-181c in neuronal morphology, and provide a framework of understanding how dysregulation of a neurodevelopmentally relevant miR in the amygdala may contribute to the pathophysiology of ASD.

MicroRNA-338 Attenuates Cortical Neuronal Outgrowth by Modulating the Expression of Axon Guidance Genes

MicroRNAs (miRs) are small non-coding RNAs that confer robustness to gene networks through post-transcriptional gene regulation. Previously, we identified miR-338 as a modulator of axonal outgrowth in sympathetic neurons. In the current study, we examined the role of miR-338 in the development of cortical neurons and uncovered its downstream mRNA targets. Long-term inhibition of miR-338 during neuronal differentiation resulted in reduced dendritic complexity and altered dendritic spine morphology. Furthermore, monitoring axon outgrowth in cortical cells revealed that miR-338 overexpression decreased, whereas inhibition of miR-338 increased axonal length. To identify gene targets mediating the observed phenotype, we inhibited miR-338 in cortical neurons and performed whole-transcriptome analysis. Pathway analysis revealed that miR-338 modulates a subset of transcripts involved in the axonal guidance machinery by means of direct and indirect gene targeting. Collectively, our results implicate miR-338 as a novel regulator of cortical neuronal maturation by fine-tuning the expression of gene networks governing cortical outgrowth.

MicroRNA-181 promotes synaptogenesis and attenuates axonal outgrowth in cortical neurons

MicroRNAs (miRs) are non-coding gene transcripts abundantly expressed in both the developing and adult mammalian brain. They act as important modulators of complex gene regulatory networks during neuronal development and plasticity. miR-181c is highly abundant in cerebellar cortex and its expression is increased in autism patients as well as in an animal model of autism. To systematically identify putative targets of miR-181c, we repressed this miR in growing cortical neurons and found over 70 differentially expressed target genes using transcriptome profiling. Pathway analysis showed that the miR-181c-modulated genes converge on signaling cascades relevant to neurite and synapse developmental processes. To experimentally examine the significance of these data, we inhibited miR-181c during rat cortical neuronal maturation in vitro; this loss-of miR-181c function resulted in enhanced neurite sprouting and reduced synaptogenesis. Collectively, our findings suggest that miR-181c is a modulator of gene networks associated with cortical neuronal maturation.

The Multifarious Hippocampal Functions of MicroRNA-137

MicroRNAs (miRs) have emerged as a powerful class of endogenous noncoding RNAs involved in posttranscriptional gene expression regulation. miR-137 has repeatedly been associated with schizophrenia and intellectual disability. Recent studies describe the mechanisms of miR-137 in mediating basic synaptic transmission and plasticity in the hippocampus. A picture is emerging in which miR-137 acts as a potent player in regulating glutamatergic synaptic transmission in the hippocampus by controlling the translation of functionally critical genes at spatially opposite ends of the synapse, contributing to the pathogenesis of cognitive impairments as seen in neurodevelopmental disorders.

Recent Developments in Optical Neuromodulation Technologies

The emergence of optogenetics technology facilitated widespread applications for interrogation of complex neural networks, such as activation of specific axonal pathways, previously found impossible with electrical stimulation. Consequently, within the short period of its application in neuroscience research, optogenetics has led to findings of significant importance both during normal brain function as well as in disease. Moreover, the optimization of optogenetics for in vivo studies has allowed the control of certain behavioral responses such as motility, reflex, and sensory responses, as well as more complex emotional and cognitive behaviors such as decision-making, reward seeking, and social behavior in freely moving animals. These studies have produced a wide variety of animal models that have resulted in fundamental findings and enhanced our understanding of the neural networks associated with behavior. The increasing number of opsins available for this technique enabled even broader regulation of neuronal activity. These advancements highlight the potential of this technique for future treatment of human diseases. Here, we provide an overview of the recent developments in the field of optogenetics technology that are relevant for a better understanding of several neuropsychiatric and neurodegenerative disorders and may pave the way for future therapeutic interventions.