Zheng Li - US grants

We have investigated the subcellular localization and effect of overexpressing neuregulin in hippocampal neurons. Our previous studies suggest that neuregulin exists in clusters in hippocampal neurons. We examined whether these neuregulin clusters associate with specific subcellular regions, and whether the pattern of distribution is regulated during neuronal development. We stained neuorns transfected with a plasmid expressing neuregulin with antibodies against synaptic proteins. We did not detect significant colocalization of neuregulin with synaptic markers. Treatments that enhance or decrease neuronal activities did not affect the subcellular localization of neuregulin. Overexpression of neuregulin altered the morphological development of synapses. This finding indicates that neuregulin regulates synapse development. Therefore neuregulin might contribute to developmental deficits of neural networks underlying schizophrenia.

miRNAs regulate protein synthesis by suppressing translation or destabilizing mRNAs. Their role in development and function of the nervous system is not well understood. This year, we identified a miRNA that regulates the development and function of synapses in the hippocampus. We have also determined the target mRNA that is regulated by this miRNA and demonstrated that the expression level of the target mRNA is inversely correlated with the identified miRNA. In addition, using overexpression and knockdown approaches, we show that the regulation of the miRNA on synapse development is mediated by its target mRNA. These findings indicate that miRNAs play important roles in the development of synapses.

1. The function of miRNAs in long-term synaptic potentiation. Long-term potentiation (LTP) of synaptic transmission is a form of synaptic plasticity that leads to long-lasting enhancement of synaptic strength. LTP is a prominent cellular model for encoding and storing information in the brain. On the basis of temporal characteristics, LTP is classified into two forms: short- and long-lasting LTP. Short-lasting LTP is induced by weak stimulation of synaptic inputs, persists for no more than 2 hours and involves no protein synthesis. Long-lasting LTP, which is induced by strong stimulation, lasts longer than 2 hours, requires de novo protein synthesis and is essential for memory formation. Little is yet known about the mechanisms of gene-specific regulation of translation during LTP. In this project, we combine next-generation sequencing, bioinformatics, electrophysiology and time-lapse imaging to investigate the role of miRNAs in LTP. These studies lead to the identification of miR-26a, miR-384-5p and let-7a as essential for LTP maintenance and enlargement of dendritic spines. Moreover, we show that ribosomal S6 kinase 3 (RSK3) mediates the function of miR-26a and miR-384-5p in LTP and present computational evidence that miRNAs exert both diverse and concerted effects in LTP. 2. miRNAs mediate activity-dependent regulation of AMPA receptors. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ionotropic glutamate receptors that mediate fast excitatory synaptic transmission in the central nervous system. The number of AMPARs in synapses determines the strength of synaptic transmission, and their abnormal expression has been implicated in cognitive impairments associated with such neurological and neuropsychiatric diseases as Alzheimers Disease, ischemia, schizophrenia, and depression. AMPAR expression is regulated by synaptic activity. During activity-dependent synaptic plasticity, for instance, the activation of N-methyl-d-aspartate (NMDA) or metabotropic glutamate receptors affects the abundance of synaptic AMPAR through both posttranslational mechanisms (including phosphorylation, palmitoylation, and ubiquitination) and local translation of dendritic mRNAs encoding AMPAR subunits. Activity-dependent modulation of AMPAR is an important mechanism that tunes synaptic strength to refine synaptic connectivity during brain development and to store information in the brain during learning and memory. Despite the broad recognition that AMPARs play a pivotal role in brain functions, molecular mechanisms underlying their regulation, especially activity-dependent local translation of AMPARs in dendrites, are only incompletely understood. In this project, we tested whether or not miRNAs regulate AMPA receptor expression in an activity-dependent manner. We combined miRNA pull-down and computational prediction to search for miRNAs that target mRNAs encoding the AMPAR subunit GluA1. This approach leads to the identification of miR-501-3p as an AMPA receptor-targeting miRNA. Our further analysis of miR-501-3p shows that it is increased locally in dendrites after NMDAR activation and that this up-regulation of miR-501-3p is required for NMDAR-dependent inhibition of AMPA receptor expression, long-lasting spine shrinkage, and elimination. These findings reveal that miRNAs are important regulators of activity-dependent local synthesis of dendritic AMPARs. 3. The change in miRNA expression in schizophrenia brains. miRNAs have been implicated in schizophrenia. Mutations in genes encoding miRNAs or components in the miRNA biogenesis machinery are associated with increased risk for schizophrenia. Using microarray and quantitative PCR, several groups have examined miRNA expression in postmortem brains of schizophrenia patients, and consistently detected miRNA expression change. Microarray and PCR-based methods, however, are constrained by their reproducibility of quantitation due to their limited sensitivity and specificity, and variable and complicated normalization methods. Here, we employed the next-generation sequencing technology, which has superior sensitivity and quantifiability, to delineate the miRNAome in the DLPFC of schizophrenia patients. We identified miRNAs that are differentially expressed in schizophrenic brains. Using bioinformatics, we found that the targets of these miRNAs are enriched for synaptic genes, which are known to be dysregulated in schizophrenia patients. Hence, miRNAs may contribute to the synaptopathology of schizophrenia. This possibility is further supported by our finding that most miRNAs altered in schizophrenia cases are expressed predominantly in one specific period from fetus to child in unaffected people when synaptic connections are formed and refined.