Developmental and Activity-Dependent miRNA Expression Profiling in Primary Hippocampal Neuron Cultures

MicroRNAs (miRNAs) are evolutionarily conserved non-coding RNAs of ∼22 nucleotides that regulate gene expression at the level of translation and play vital roles in hippocampal neuron development, function and plasticity. Here, we performed a systematic and in-depth analysis of miRNA expression profiles in cultured hippocampal neurons during development and after induction of neuronal activity. MiRNA profiling of primary hippocampal cultures was carried out using locked nucleic-acid-based miRNA arrays. The expression of 264 different miRNAs was tested in young neurons, at various developmental stages (stage 2-4) and in mature fully differentiated neurons (stage 5) following the induction of neuronal activity using chemical stimulation protocols. We identified 210 miRNAs in mature hippocampal neurons; the expression of most neuronal miRNAs is low at early stages of development and steadily increases during neuronal differentiation. We found a specific subset of 14 miRNAs with reduced expression at stage 3 and showed that sustained expression of these miRNAs stimulates axonal outgrowth. Expression profiling following induction of neuronal activity demonstrates that 51 miRNAs, including miR-134, miR-146, miR-181, miR-185, miR-191 and miR-200a show altered patterns of expression after NMDA receptor-dependent plasticity, and 31 miRNAs, including miR-107, miR-134, miR-470 and miR-546 were upregulated by homeostatic plasticity protocols. Our results indicate that specific miRNA expression profiles correlate with changes in neuronal development and neuronal activity. Identification and characterization of miRNA targets may further elucidate translational control mechanisms involved in hippocampal development, differentiation and activity-depended processes

Corticosterone Alters AMPAR Mobility and Facilitates Bidirectional Synaptic Plasticity

Background: The stress hormone corticosterone has the ability both to enhance and suppress synaptic plasticity and learning and memory processes. However, until today there is very little known about the molecular mechanism that underlies the bidirectional effects of stress and corticosteroid hormones on synaptic efficacy and learning and memory processes. In this study we investigate the relationship between corticosterone and AMPA receptors which play a critical role in activity-dependent plasticity and hippocampal-dependent learning. Methodology/Principal Findings: Using immunocytochemistry and live cell imaging techniques we show that corticosterone selectively increases surface expression of the AMPAR subunit GluR2 in primary hippocampal cultures via a glucocorticoid receptor and protein synthesis dependent mechanism. In agreement, we report that corticosterone also dramatically increases the fraction of surface expressed GluR2 that undergo lateral diffusion. Furthermore, our data indicate that corticosterone facilitates NMDAR-invoked endocytosis of both synaptic and extra-synaptic GluR2 under conditions that weaken synaptic transmission. Conclusion/Significance: Our results reveal that corticosterone increases mobile GluR2 containing AMPARs. The enhanced lateral diffusion properties can both facilitate the recruitment of AMPARs but under appropriate conditions facilitate the loss of synaptic AMPARs (LTD). These actions may underlie both the facilitating and suppressive effects of corticosteroid hormones on synaptic plasticity and learning and memory and suggest that these hormones accentuate synaptic efficacy

Synapse Pathology in Psychiatric and Neurologic Disease

Inhibitory and excitatory synapses play a fundamental role in information processing in the brain. Excitatory synapses usually are situated on dendritic spines, small membrane protrusions that harbor glutamate receptors and postsynaptic density components and help transmit electrical signals. In recent years, it has become evident that spine morphology is intimately linked to synapse function—smaller spines have smaller synapses and support reduced synaptic transmission. The relationship between synaptic signaling, spine shape, and brain function is never more apparent than when the brain becomes dysfunctional. Many psychiatric and neurologic disorders, ranging from mental retardation and autism to Alzheimer’s disease and addiction, are accompanied by alterations in spine morphology and synapse number. In this review, we highlight the structure and molecular organization of synapses and discuss functional effects of synapse pathology in brain disease

Developmental and Activity-Dependent miRNA Expression Profiling in Primary Hippocampal Neuron Cultures

MicroRNAs (miRNAs) are evolutionarily conserved non-coding RNAs of similar to 22 nucleotides that regulate gene expression at the level of translation and play vital roles in hippocampal neuron development, function and plasticity. Here, we performed a systematic and in-depth analysis of miRNA expression profiles in cultured hippocampal neurons during development and after induction of neuronal activity. MiRNA profiling of primary hippocampal cultures was carried out using locked nucleic-acid-based miRNA arrays. The expression of 264 different miRNAs was tested in young neurons, at various developmental stages (stage 2-4) and in mature fully differentiated neurons (stage 5) following the induction of neuronal activity using chemical stimulation protocols. We identified 210 miRNAs in mature hippocampal neurons; the expression of most neuronal miRNAs is low at early stages of development and steadily increases during neuronal differentiation. We found a specific subset of 14 miRNAs with reduced expression at stage 3 and showed that sustained expression of these miRNAs stimulates axonal outgrowth. Expression profiling following induction of neuronal activity demonstrates that 51 miRNAs, including miR-134, miR-146, miR-181, miR-185, miR-191 and miR-200a show altered patterns of expression after NMDA receptor-dependent plasticity, and 31 miRNAs, including miR-107, miR-134, miR-470 and miR-546 were upregulated by homeostatic plasticity protocols. Our results indicate that specific miRNA expression profiles correlate with changes in neuronal development and neuronal activity. Identification and characterization of miRNA targets may further elucidate translational control mechanisms involved in hippocampal development, differentiation and activity-depended processes

Corticosterone induces a delayed enhancement of the mEPSC amplitude.

<p>A) mEPSC amplitude and (B) frequency after treatment with vehicle or corticosterone. C) Normalized frequency histogram for the distribution of the amplitude of mEPSCs in hippocampal primary neurons after control treatment or treatment with corticosterone. A shift toward larger amplitudes was observed after hormone treatment. D) Cumulative frequency histogram shows a marked shift toward larger amplitude mEPSCs after corticosterone treatment. ^*P<0.05.</p

Corticosterone treatment dramatically modifies the endocytic properties of AMPARs.

<p>A) Rapid endocytosis of synaptic (punctate) and extrasynaptic (diffuse) AMPARs induced by activation of NMDARs in control hippocampal neurons. Scale bar, 20 µm. B) Rapid endocytosis of synaptic (punctate) and extrasynaptic (diffuse) AMPARs induced by activation of NMDARs in corticosterone-treated hippocampal neurons. Scale bar, 20 µm. C) Binned and averaged fluorescence values from punctate (red) and diffuse (blue) regions during and after NMDAR stimulation in control treated cells. Data reflect error bars show±SEM (4 cells for each condition with 14 punctate and 17 diffuse regions). D) Binned and averaged fluorescence values from punctate (red) and diffuse (blue) regions during and after NMDAR stimulation in corticosterone treated cells. Data reflect error bars show±SEM (4 cells for each condition with 14 punctate and 17 diffuse regions).</p

Glucocorticoid receptor activation promotes surface AMPA receptor expression.

<p>A) Representative images of hippocampal neurons at DIV13 treated with vehicle, 30 nM and 100 nM corticosterone for 3 h and stained for surface GluR1 (red) and GluR2 (green). B) Quantification of GluR1 and GluR2 intensity after treatment with vehicle and 0.3–100 nM corticosterone for 3 h. C) Quantification of surface GluR1 and GluR2 intensity after treatment with vehicle for 3 h and 30 nM corticosterone for 1 or 3 h. In addition cells were incubated for 3 h with CORT, washed and incubated in regular medium for 21 h (3 h+21 h). D) Quantification of total GluR1 and GluR2 intensity after treatment with vehicle and 30 nM corticosterone for 3 h. E) Quantification of surface GluR1 and GluR2 intensity of primary hippocampal neurons. Cells were treated with vehicle, 100 µM cycloheximide or 500 nM RU 38486 for 3 h followed 30 min later with vehicle or 30 nM corticosterone applications for 3 h. F) Representative Western blots show expression of GluR1, GluR2, transferrin receptor (TrfR), actin and tubulin in total and surface fraction of biotinylated primary hippocampal cultures treated with vehicle (−) or 100 nM corticosterone (+) for 3 h. G) Quantitative analysis of surface expression of GluR1, GluR2 and transferrin receptor (TrfR) in biotinylated primary hippocampal neurons treated with 100 nM corticosterone for 3 h. H) Quantitative analysis of total expression of GluR1, GluR2, transferrin receptor (TrfR), actin and tubulin in biotinylated primary hippocampal neurons treated with 100 nM corticosterone for 3 h.</p

AP-2 levels in primary hippocampal neurons treated with corticosterone.

<p>Hippocampal neurons (DIV 22) were treated with vehicle (lane 1–4) or 100 nM corticosterone (lane 5–8) for 3 h and harvested directly in sample buffer. The immunoblots were probed with two different anti-AP-2 (from Sigma and BD biosciences), anti-α-tubulin and anti-pan-actin antibodies. The positions of molecular weight standards (kDa) are indicated at left. No difference in AP-2 expression levels between control and corticosterone treated neurons is observed.</p

TRAK/Milton motor-adaptor proteins steer mitochondrial trafficking to axons and dendrites

In neurons, the distinct molecular composition of axons and dendrites is established through polarized targeting mechanisms, but it is currently unclear how nonpolarized cargoes, such as mitochondria, become uniformly distributed over these specialized neuronal compartments. Here, we show that TRAK family adaptor proteins, TRAK1 and TRAK2, which link mitochondria to microtubule-based motors, are required for axonal and dendritic mitochondrial motility and utilize different transport machineries to steer mitochondria into axons and dendrites. TRAK1 binds to both kinesin-1 and dynein/dynactin, is prominently localized in axons, and is needed for normal axon outgrowth, whereas TRAK2 predominantly interacts with dynein/dynactin, is more abundantly present in dendrites, and is required for dendritic development. These functional differences follow from their distinct conformations: TRAK2 preferentially adopts a head-to-tail interaction, which interferes with kinesin-1 binding and axonal transport. Our study demonstrates how the molecular interplay between bidirectional adaptor proteins and distinct microtubule-based motors drives polarized mitochondrial transport

TRAK/Milton motor-adaptor proteins steer mitochondrial trafficking to axons and dendrites

In neurons, the distinct molecular composition of axons and dendrites is established through polarized targeting mechanisms, but it is currently unclear how nonpolarized cargoes, such as mitochondria, become uniformly distributed over these specialized neuronal compartments. Here, we show that TRAK family adaptor proteins, TRAK1 and TRAK2, which link mitochondria to microtubule-based motors, are required for axonal and dendritic mitochondrial motility and utilize different transport machineries to steer mitochondria into axons and dendrites. TRAK1 binds to both kinesin-1 and dynein/dynactin, is prominently localized in axons, and is needed for normal axon outgrowth, whereas TRAK2 predominantly interacts with dynein/dynactin, is more abundantly present in dendrites, and is required for dendritic development. These functional differences follow from their distinct conformations: TRAK2 preferentially adopts a head-to-tail interaction, which interferes with kinesin-1 binding and axonal transport. Our study demonstrates how the molecular interplay between bidirectional adaptor proteins and distinct microtubule-based motors drives polarized mitochondrial transport