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.

The release of apoptotic molecules from mitochondria is activated by the pro-apoptotic Bcl-2 proteins in programmed cell death. We first ask whether the pro-apoptotic Bcl-2 proteins activate mitochondria in long-term depression (LTD). We will alter the expression of pro-apoptotic Bcl-2 proteins in hippocampal neurons, then examine -amino-3-hydroxy-5-methylisoxazole-4- propionic acid (AMPA) receptor endocytosis and LTD in these neurons. To investigate how casapses mediate AMPA receptor endocytosis, we will use antibodies against caspase-3 to immunoprecipitate proteins that bind to caspase-3 in LTD. We have made a construct to overexpress caspase-3 in hippocampal neurons and are performing the immunoprecipitation experiment.

This year, we revealed a relationship between long-term depression (LTD), mitochondria and caspases. In this study we show that activation of caspase-3 through mitochondria is actually required for long-term depresssion (LTD) and for the internalization of AMPA receptor GluR2 in mouse hippocampal neurons. We report that regulatory factors and executors of apoptosis play a role in the molecular mechanism of synaptic plasticity. We closely examined the mitochondrial pathway, which is mediated by caspase-9 and caspase-3, as being an essential part of LTD but not LTP. We show that caspase-3 knockout hippocampal slices are defective in LTD while LTP remains unaffected. This is compelling evidence for the specificity of caspase-3 in one form of synaptic modification. The importance of the mitochondrial signaling pathway in LTD is related with its critical role in inducible GluR2 AMPA receptor internalization. Caspases-3 and -9 are crucial for LTD and GluR2 trafficking, but not for LTP. In addition, short-term glutamate receptor stimulation sufficient to induce AMPA receptor redistribution and synaptic modification causes caspase-3 activation that is transient and mild, as compared with a conventional factor that cause massive apoptosis. This mild activation of caspase-3 by short-duration glutamate receptor stimulation was unrelated with increased cell death, a strong indicator that caspase-3 does not inevitably lead to apoptosis. It is believed that the complex morphology of neurons could allow for localized activation of caspases, so that spines and dendrites could be targeted for functional attrition and structural elimination without killing the rest of the cell.

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 molecular mechanism underlying long-term synaptic depression. The strength of synaptic transmission can change through synaptic plasticity. Long-lasting forms of synaptic plasticity (such as long-term potentiation and long-term depression of synaptic transmission) are important cellular mechanisms underlying information storage in the brain and the establishment of proper neural circuits during development. In this project, we investigated the mechanism underlying the induction of long-term depression of synaptic transmission (LTD). Our group has found that macroautophagy(autophagy hereinafter) plays an important role in LTD. Autophagy is a cellular process which mediates the degradation of cytoplasmic components and organelles via lysosomes. . Autophagy is also essential for the development and function of synapses. It enables developmental pruning of dendritic spines (subcellular structures accommodating postsynaptic components), and regulates presynaptic structure, dopamine release, and degradation of postsynaptic receptors. Anomalous autophagy is associated with brain disorders. Autophagy is orchestrated by more than 30 autophagy-related (Atg) proteins and multiple signaling pathways. Mechanistic target of rapamycin complex 1 (mTORC1) is the best-characterized regulator of autophagy induction in mammalian cells. Using mTOR inhibitors and knockout mice with deficient autophagy, we previously found that autophagic flux changes during LTD and this in turn leads to AMPA receptor endocytosis. During this reporting period, we investigated the mechanism by which autophagy regulates AMPA receptor internalization. We also examined the effect of altering autophagy on the behavior of mice. 2. The role of dysbindin-1 in synaptic physiology. Dysbindin-1 is a coiled-coil domain containing protein, initially discovered as a dystrophin-binding protein and later found to be one of eight subunits of biogenesis of lysosome-related organelles complex 1 (BLOC-1). The postmortem brains of individuals with schizophrenia consistently exhibit low levels of dysbindin-1 proteins and mRNAs. Our earlier work shows that dysbindin-1 contributes to the establishment of neuronal connectivity by regulating the growth of dendritic protrusions, including dendritic spines (tiny dendritic protrusions where excitatory synapses are formed) and filopodia (long, thin protrusions that serve as precursors of dendritic spines in young neurons). Dysbindin-1, therefore, may confer the risk for schizophrenia by regulating the development of dendritic spines. During this review period, we investigated the role of dysbindin-1 in psychogenic stress-induced synaptic alterations. We found that synaptic plasticity is more sensitive to psychogenic stress in dysbindin-1 mutant mice than in wild-type mice. Mild stress does not significantly affect synaptic plasticity in wild-type mice but alters social behavior in dysbindin-1 mutant mice. We recorded in the brain slice of stressed mice and found that stress alters synaptic physiology in mutant but not in wild-type mice. During this reporting period, we investigated the mechanism underlying this phenomenon and focused on determining the type of neurotransmitter receptors involved in psychogenic stress-induced synaptic alterations.

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.