Giovanni Lugli

Dicer and eIF2c are enriched at postsynaptic densities in adult mouse brain and are modified by neuronal activity in a calpain-dependent manner.

We have hypothesized that small RNAs may participate in learning and memory mechanisms. Because dendritic spines are important in synaptic plasticity and learning, we asked whether dicer, the rate‐limiting enzyme in the formation of small RNAs, is enriched within dendritic spines. In adult mouse brain, dicer and the RNA‐induced silencing complex (RISC) component eIF2c were expressed in the somatodendritic compartment of principal neurons and some interneurons in many regions, and dicer was enriched in dendritic spines and postsynaptic densities (PSDs). A portion of dicer and eIF2c were associated with each other and with fragile X mental retardation protein (FMRP), as assessed by co‐immunoprecipitation. Calpain I treatment of recombinant dicer or immunopurified brain dicer caused a marked increase in RNAse III activity. Purified PSDs did not exhibit RNAse III activity, but calpain caused release of dicer from PSDs in an enzymatically active form, together with eIF2c. NMDA stimulation of hippocampal slices, or calcium treatment of synaptoneurosomes, caused a 75 kDa dicer fragment to appear in a calpain‐dependent manner. The findings support a model whereby acute neuronal stimulation at excitatory synapses increases intracellular calcium, which activates calpain, which liberates dicer and eIF2c bound to PSDs. This supports the hypothesis that dicer could be involved in synaptic plasticity.

Expression of microRNAs and their precursors in synaptic fractions of adult mouse forebrain

We have characterized the expression of microRNAs and selected microRNA precursors within several synaptic fractions of adult mouse forebrain, including synaptoneurosomes, synaptosomes and isolated post‐synaptic densities (PSDs), using methods of microRNA microarray, real time qRT‐PCR, Northern blotting and immunopurification using anti‐PSD95 antibody. The majority of brain microRNAs (especially microRNAs known to be expressed in pyramidal neurons) are detectably expressed in synaptic fractions, and a subset of microRNAs is significantly enriched in synaptic fractions relative to total forebrain homogenate. MicroRNA precursors are also detectable in synaptic fractions at levels that are comparable to whole tissue. Whereas mature microRNAs are predominantly associated with soluble components of the synaptic fractions, microRNA precursors are predominantly associated with PSDs. For seven microRNAs examined, there was a significant correlation between the relative synaptic enrichment of the precursor and the relative synaptic enrichment of the corresponding mature microRNA. These findings support the proposal that microRNAs are formed, at least in part, via processing of microRNA precursors locally within dendritic spines. Dicer is expressed in PSDs but is enzymatically inactive until conditions that activate calpain cause its liberation; thus, we propose that synaptic stimulation may lead to local processing of microRNA precursors in proximity to the synapse.

MicroRNA Expression Is Down-Regulated and Reorganized in Prefrontal Cortex of Depressed Suicide Subjects

Background Recent studies suggest that alterations in expression of genes, including those which regulate neural and structural plasticity, may be crucial in the pathogenesis of depression. MicroRNAs (miRNAs) are newly discovered regulators of gene expression that have recently been implicated in a variety of human diseases, including neuropsychiatric diseases. Methodology/Principal Findings The present study was undertaken to examine whether the miRNA network is altered in the brain of depressed suicide subjects. Expression of miRNAs was measured in prefrontal cortex (Brodmann Area 9) of antidepressant-free depressed suicide (n = 18) and well-matched non-psychiatric control subjects (n = 17) using multiplex RT-PCR plates. We found that overall miRNA expression was significantly and globally down-regulated in prefrontal cortex of depressed suicide subjects. Using individual tests of statistical significance, 21 miRNAs were significantly decreased at p = 0.05 or better. Many of the down-regulated miRNAs were encoded at nearby chromosomal loci, shared motifs within the 5′-seeds, and shared putative mRNA targets, several of which have been implicated in depression. In addition, a set of 29 miRNAs, whose expression was not pairwise correlated in the normal controls, showed a high degree of co-regulation across individuals in the depressed suicide group. Conclusions/Significance The findings show widespread changes in miRNA expression that are likely to participate in pathogenesis of major depression and/or suicide. Further studies are needed to identify whether the miRNA changes lead to altered expression of prefrontal cortex mRNAs, either directly (by acting as miRNA targets) or indirectly (e.g., by affecting transcription factors).

microRNA Regulation of Synaptic Plasticity

AbstractmicroRNAs play an important role in regulating synaptic plasticity. For example, microRNAs target (and are targeted by) plasticity mediators such as CREB, MECP2, and FMRP. As well, specific microRNAs have been shown to be expressed within dendrites, where they regulate protein translation of targets mediating dendritic growth. Components of the RISC machinery have been implicated in long-term memory in Drosophila. Here, we review evidence from studies of adult mouse forebrain supporting a model wherein synaptic stimulation (above a threshold value) increases calcium within dendritic spines, activates calpain, and activates and releases dicer from the postsynaptic density. Dicer processes local pre-miRs into mature miRNAs that are incorporated into RISC complexes within or near the dendritic spine, and that bind available target mRNAs in the vicinity. These may repress protein translation under resting conditions, yet permit a phasic burst of translation to occur transiently following subsequent synaptic activity. Loaded RISC complexes that are not bound to local mRNAs may serve to bind and trap mRNAs that are being transported down dendrites. Thus, locally formed microRNAs may mark the location of previously activated synapses and perform a type of synaptic tagging and capture.

Expression of microRNAs and Other Small RNAs in Prefrontal Cortex in Schizophrenia, Bipolar Disorder and Depressed Subjects

Because of the role played by miRNAs in post-transcriptional regulation of an array of genes, their impact in neuropsychiatric disease pathophysiology has increasingly been evident. In the present study, we assessed microRNA expression in prefrontal cortex (Brodmann area 10) of a well-characterized cohort of major depressed, bipolar, and schizophrenia subjects (obtained from Stanley Neuropathology Consortium; n = 15 in each group), using high throughput RT-PCR plates. Discrete miRNA alterations were observed in all disorders, as well as in suicide subjects (pooled across diagnostic categories) compared to all non-suicide subjects. The changes in the schizophrenia group were partially similar to those in the bipolar group, but distinct from changes in depression and suicide. Intriguingly, those miRNAs which were down-regulated in the schizophrenia group tended to be synaptically enriched, whereas up-regulated miRNAs tended not to be. To follow this up, we purified synaptosomes from pooled samples of the schizophrenia vs. control groups and subjected them to Illumina deep sequencing. There was a significant loss of small RNA expression in schizophrenia synaptosomes only for certain sequence lengths within the miRNA range. Moreover, 73 miRNAs were significantly down-regulated whereas only one was up-regulated. Strikingly, across all expressed miRNAs in synaptosomes, there was a significant inverse correlation between the fold-change of a given miRNA seen in schizophrenia and its synaptic enrichment ratio observed in controls. Thus, synaptic miRNAs tended to be down-regulated in schizophrenia, and the more highly synaptically enriched miRNAs tended to show greater down-regulation. These findings point to some deficit in miRNA biogenesis, transport, processing or turnover in schizophrenia that is selective for the synaptic compartment. A novel class of ncRNA-derived small RNAs, shown to be strongly induced during an early phase of learning in mouse, is also expressed in man, and at least one representative (SNORD85) was strongly down-regulated in schizophrenia synaptosomes.

Plasma Exosomal miRNAs in Persons with and without Alzheimer Disease: Altered Expression and Prospects for Biomarkers

To assess the value of exosomal miRNAs as biomarkers for Alzheimer disease (AD), the expression of microRNAs was measured in a plasma fraction enriched in exosomes by differential centrifugation, using Illumina deep sequencing. Samples from 35 persons with a clinical diagnosis of AD dementia were compared to 35 age and sex matched controls. Although these samples contained less than 0.1 microgram of total RNA, deep sequencing gave reliable and informative results. Twenty miRNAs showed significant differences in the AD group in initial screening (miR-23b-3p, miR-24-3p, miR-29b-3p, miR-125b-5p, miR-138-5p, miR-139-5p, miR-141-3p, miR-150-5p, miR-152-3p, miR-185-5p, miR-338-3p, miR-342-3p, miR-342-5p, miR-548at-5p, miR-659-5p, miR-3065-5p, miR-3613-3p, miR-3916, miR-4772-3p, miR-5001-3p), many of which satisfied additional biological and statistical criteria, and among which a panel of seven miRNAs were highly informative in a machine learning model for predicting AD status of individual samples with 83–89% accuracy. This performance is not due to over-fitting, because a) we used separate samples for training and testing, and b) similar performance was achieved when tested on technical replicate data. Perhaps the most interesting single miRNA was miR-342-3p, which was a) expressed in the AD group at about 60% of control levels, b) highly correlated with several of the other miRNAs that were significantly down-regulated in AD, and c) was also reported to be down-regulated in AD in two previous studies. The findings warrant replication and follow-up with a larger cohort of patients and controls who have been carefully characterized in terms of cognitive and imaging data, other biomarkers (e.g., CSF amyloid and tau levels) and risk factors (e.g., apoE4 status), and who are sampled repeatedly over time. Integrating miRNA expression data with other data is likely to provide informative and robust biomarkers in Alzheimer disease.

Endogenous siRNAs and noncoding RNA-derived small RNAs are expressed in adult mouse hippocampus and are up-regulated in olfactory discrimination training.

We previously proposed that endogenous siRNAs may regulate synaptic plasticity and long-term gene expression in the mammalian brain. Here, a hippocampal-dependent task was employed in which adult mice were trained to execute a nose-poke in a port containing one of two simultaneously present odors in order to obtain a reward. Mice demonstrating olfactory discrimination training were compared to pseudo-training and nose-poke control groups; size-selected hippocampal RNA was subjected to Illumina deep sequencing. Sequences that aligned uniquely and exactly to the genome without uncertain nucleotide assignments, within exons or introns of MGI annotated genes, were examined further. The data confirm that small RNAs having features of endogenous siRNAs are expressed in brain; that many of them derive from genes that regulate synaptic plasticity (and have been implicated in neuropsychiatric diseases); and that hairpin-derived endo-siRNAs and the 20- to 23-nt size class of small RNAs show a significant increase during an early stage of training. The most abundant putative siRNAs arose from an intronic inverted repeat within the SynGAP1 locus; this inverted repeat was a substrate for dicer in vitro, and SynGAP1 siRNA was specifically associated with Argonaute proteins in vivo. Unexpectedly, a dramatic increase with training (more than 100-fold) was observed for a class of 25- to 30-nt small RNAs derived from specific sites within snoRNAs and abundant noncoding RNAs (Y1 RNA, RNA component of mitochondrial RNAse P, 28S rRNA, and 18S rRNA). Further studies are warranted to characterize the role(s) played by endogenous siRNAs and noncoding RNA-derived small RNAs in learning and memory.

Natural antisense transcripts are co-expressed with sense mRNAs in synaptoneurosomes of adult mouse forebrain

Natural antisense transcripts and overlapping sense transcripts are expressed in a variety of tissues, including adult mouse brain. Here we show that a subset of mRNA-like sense-antisense transcript pairs are co-expressed within synaptoneurosomes of adult mouse forebrain, a subcellular fraction that is enriched in pinched-off dendritic spines of pyramidal neurons. Several of these pairs involve mRNAs that have been implicated in synaptic functions and in Alzheimer disease pathways. This study provides evidence that a new class of noncoding RNAs (natural antisense transcripts) are expressed near synapses, and encourages further studies of their roles in neuronal function.

Primary microRNA precursor transcripts are localized at post-synaptic densities in adult mouse forebrain.

In a previous study, we reported that microRNA (miRNA) precursors are expressed in synaptic fractions within adult mouse forebrain, where they are enriched at post‐synaptic densities (PSDs). However, because that study employed qRT‐PCR primers that recognize the hairpin region, it was not able to distinguish between primary microRNA gene transcripts (pri‐miRs) and small hairpin precursors (pre‐miRs). Here, using primer sets that selectively measure regions upstream, downstream and flanking the hairpin, we demonstrate that pri‐miRs are present in synaptic fractions (enriched several‐fold relative to total tissue homogenate) and are especially enriched in isolated PSDs. Drosha and DGCR8 proteins are also expressed in synaptic fractions and PSDs, and are tightly associated with pri‐miRs as assessed by coimmunoprecipitation under stringent conditions. Pri‐miRs, drosha, and DGCR8 are highly enriched in fractions that contain mRNA transport particles, and cytosolic drosha is associated with kinesin heavy chain; these findings suggest that pri‐miRs are transported to synaptic regions in a manner similar to mRNAs. This study supports the notion that miRNA biogenesis occurs locally near synapses in a regulated fashion.

Olfactory discrimination training up-regulates and reorganizes expression of microRNAs in adult mouse hippocampus

Adult male mice (strain C57Bl/6J) were trained to execute nose-poke responses for water reinforcement; then they were randomly assigned to either of two groups: olfactory discrimination training (exposed to two odours with reward contingent upon correctly responding to one odour) or pseudo-training (exposed to two odours with reward not contingent upon response). These were run in yoked fashion and killed when the discrimination-trained mouse reached a learning criterion of 70% correct responses in 20 trials, occurring after three sessions (a total of ∼40 min of training). The hippocampus was dissected bilaterally from each mouse (N = 7 in each group) and profiling of 585 miRNAs (microRNAs) was carried out using multiplex RT–PCR (reverse transcription–PCR) plates. A significant global up-regulation of miRNA expression was observed in the discrimination training versus pseudo-training comparison; when tested individually, 29 miRNAs achieved significance at P = 0.05. miR-10a showed a 2.7-fold increase with training, and is predicted to target several learning-related mRNAs including BDNF (brain-derived neurotrophic factor), CAMK2b (calcium/calmodulin-dependent protein kinase IIβ), CREB1 (cAMP-response-element-binding protein 1) and ELAVL2 [ELAV (embryonic lethal, abnormal vision, Drosophila)-like; Hu B]. Analysis of miRNA pairwise correlations revealed the existence of several miRNA co-expression modules that were specific to the training group. These in vivo results indicate that significant, dynamic and co-ordinated changes in miRNA expression accompany early stages of learning.