Andres Buonanno, PhD - US grants
Affiliations: | Developmental Neurobiology | National Institutes of Health, Bethesda, MD |
Area:
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According to our matching algorithm, Andres Buonanno is the likely recipient of the following grants.Years | Recipients | Code | Title / Keywords | Matching score |
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2007 — 2011 | Buonanno, Andres | Z01Activity Code Description: Undocumented code - click on the grant title for more information. ZIAActivity Code Description: Undocumented code - click on the grant title for more information. |
Mechanisms Regulating Activity Dependent Synaptic Plasticity and Gene Expression @ Child Health and Human Development A) NEUREGULIN/ERB-B SIGNALING REGULATES NEURONAL PLASTICITY: POSSIBLE RELEVANCE TO SCHIZOPRENIA[unreadable] [unreadable] 1. Isolation and Characterization of the Neuregulin-1 Type IV Isoform: Accumulating evidence supports the involvement of NRG-1 and ErbB-4 receptors in the etiology of schizophrenia. The NRG-1 gene generates numerous transcripts by using different transcriptional promoters and alternative splicing. Interestingly, a single nucleotide polymorphism (SNP8NRG243177) is located close to a region proposed to function as a promoter for the novel NRG-1 isoform denoted as Type IV. The SNP8NRG243177 T/T polymorphism is associated with higher levels of Type IV transcripts in postmortem tissue. Moreover, SNP8NRG243177 T/T, which maps within the previously identified schizophrenia at-risk haplotype, is associated with lower prefrontal activation and the development of psychotic symptoms. NRG-1 Type IV transcripts were originally identified by RT-PCR as partial RNA fragments. Therefore, it currently is not known whether these partial transcripts originated from full-length NRG-1 mRNAs, and whether these mRNAs encode for pro-NRG-1 proteins that are post-translationally processed to produce a biologically active form of NRG-1. Toward understanding a possible role of Type IV NRG-1 in the human brain, we isolated two full-length mRNAs encoding Type IV proteins. We found that these transcripts are translated to generate pro-NRG-1 type IV protein that is post-translationally processed, released from cells, and has the capacity to activate ErbB receptors and its downstream signaling pathways. This study provides the first evidence for the existence of NRG-1 Type IV protein. Experiments are in progress to determine if, and how, expression of NRG-1 Type IV protein is altered in schizophrenia.[unreadable] [unreadable] 2. Depotentiation of LTP by NRG-1: Understanding NRG/ErbB-4 signaling in the brain is important to understand how the pathway is altered in schizophrenia. We found that NRG-1 reverses ('depotentiates') long-term potentiation (LTP) at hippocampal CA1 glutamatergic synapses in an activity-dependent fashion. Inhibitors that selectively target ErbB tyrosine kinases block NRG-1-dependent depotentiation, and increase LTP levels at synapses already potentiated. Using patch clamp and cell biological techniques, we demonstrated that NRG-1 depotentiates LTP by selectively reducing AMPA, but not NMDA, receptor currents. Live imaging of hippocampal neurons transfected with AMPA receptors fused to superecliptic green fluorescent protein (seGFP), a form of GFP that only fluoresces strongly when expressed on the cell surface, indicates that NRG-1 stimulates the internalization of surface seGluR1-containing AMPA receptors. Consistent with these and our earlier findings, others have shown that NRG-1 and ErbB receptor hypomorphic mice develop normally, but have a reduction in glutamate receptor levels and manifest behavioral deficits. This novel regulation of LTP by NRG-1 has important implications for the modulation of synaptic homeostasis at glutamatergic synapses, which can affect cognition, learning and memory, and for understanding molecular mechanisms that underlie complex disorders like schizophrenia. [unreadable] [unreadable] 3. ErbB-4 Surface Clustering by PSD-95 at Inhibitory Hippocampal Neurons: To extend on our earlier work, showing that ErbB-4 directly interacts with the postsynaptic density protein PSD-95 at glutamatergic synapses, we investigated the developmental expression and trafficking the receptor. This interaction is of special interest because it may be altered in schizophrenia. Using immunofluorescence analysis in hippocampal slices and dissociated neurons in culture, we found that ErbB-4 receptors are expressed predominantly at glutamatergic synapses in GABAergic interneurons. The trafficking of ErbB-4 in cultured hippocampal neurons was investigated by surface protein biotinylation and antibody labeling of receptors in live cells. We found that ErbB-4 immunoreactivity in developing neurons precedes PSD-95 expression, with ErbB-4 cluster initially forming in the absence of, but later associating with, PSD-95-positive puncta. The surface fraction of dendritic ErbB-4 increases with age, and NRG-1 triggers its internalization in young and mature neurons. These findings enhance our understanding of the role of ErbB-4/PSD-95 protein interaction for NRG-mediated signaling at glutamatergic synapses.[unreadable] [unreadable] 4. Function of NMDA NR2C Receptor tested in cells from knockout mice: We previously reported that expression of the NR2C subunit of the NMDAR is regulated by NRG-1 in cultured organotypic slices from cerebellum. To study the function of the NR2C subunit, DNA sequences corresponding to the first 11 exons of the gene protein were removed by homologous recombination. In collaboration with the groups of Drs. Vicini and Wolfe, the NMDAR excitatory postsynaptic currents (EPSCs) were studied in solitary cerebellar neurons cultured in microislands from wild-type (WT) and NR2C-Bgal knock-in mice, as well as NR2A subunit knockout mice. Compared to WT cells, NR2C null granule neurons have larger NMDA-EPSCs, fast decaying currents and increased quantal content. The most striking result is a significant increase in the NMDA-EPSC peak amplitude and charge transfer in NR2C mutant cells which is mostly due to an increase in quantal size, as estimated from miniature NMDA-EPSCs. Interestingly, the protein levels of NR1, NR2A and NR2B in cerebella from 21-day-old NR2C mutant mice are decreased, as compared to wild-type, suggesting a possible compensatory response to the increased NMDA-EPSCs.[unreadable] [unreadable] B. ACTIVITY-DEPENDENT REGULATION OF MUSCLE TYPES[unreadable] [unreadable] 1. Role of activity in the adult: The effects of motor neuron electrical activity on TnI transcription were measured in vivo by imaging individual myofibers. The levels of transcription were measured in adult muscles transfected with the SURE and FIRE GFP reporter constructs before and after electrical stimulation. We found that slow, tonic depolarization up-regulated SURE transcription, while fast, phasic stimuli enhanced FIRE transcription. These results indicated that the TnI slow and fast enhancers sense, and respond to, distinct patterns of neuronal activity. [unreadable] [unreadable] 2.Identification of calcium-responsive factors: Next, we set out to identify the precise DNA elements and transcription factors that respond differentially to activity. Numerous lines of evidence indicate that the effects of activity are mediated by calcium released from the sarcoplasmic reticulum. NFAT and NFkB are two transcription factors that respond to calcium transients in T-cells to differentially regulate genes. We found that while NFAT represses transcription from FIRE in response to slow-patterned activity, NFkB increased FIRE transcription in response to fast-patterned stimuli. These experiments exemplify how muscles can modify their adult contractile properties in response to distinct types of exercise.[unreadable] [unreadable] 3. TEF3/Tead4 knockout mice: Tead4 (or TEF3) was another transcription factor analyzed for its potential role in regulating muscle development and regeneration. Tead4-floxed mice were generated by homologous recombination. Developmental studies indicated that ablation of the gene resulted in a preimplantation failure. Tead4-/- embryos do not to express trophectoderm specific genes, and the morulae do not produce trophoblast stem cells, trophectoderm, or blastocoel cavities, and therefore fail to implant. Future experiments with conditional Tead4 mutant mice will be needed to assess if Tead4 regulates fiber type properties during development. |
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2012 — 2018 | Buonanno, Andres | ZIAActivity Code Description: Undocumented code - click on the grant title for more information. |
Neuregulin-Erbb Signaling in Neuronal Development and Psychiatric Disorders @ Child Health and Human Development 1. Conservation of ErbB4 expression in GABAergic interneurons in the hippocampus and cortex, from rodents to primates: Knowledge of the cellular and subcellular localization of ErbB4 is important for understanding how Neuregulin (NRG) regulates neuronal network activity and behavior. In collaboration with Dr. McBain's and Dr. Lewis's lab, we unequivocally demonstrated, using a combination of single-cell RT-PCR and electrophysiological profiling in mice, that ErbB4 transcripts are expressed GABAergic interneurons and not in excitatory pyramidal cells. A finding confirmed with our ErbB4 mAb that showed receptor accumulates in the somato-dendritic region of cortical GABAergic interneurons in rodents, rhesus monkey and humans; presynaptic or axonal expression of ErbB4 was rarely detected. This across-species conservation validates the use of rodents to analyze cellular and neural circuit effects of abnormal ErbB4 function as a means to model endophenotypes pertinent to psychiatric disorders. 2. Importance of Neuregulin/ErbB4 signaling in parvalbumin-positive fast-spiking interneurons: We previously showed, using reverse-microdialysis neurochemistry and electrophysiology, that NRG1 signaling via ErbB4 potently triggers dopamine release in the hippocampus and reverses LTP at Schaeffer collateral-to-CA1 (SC-CA1) synapses through activation of dopamine D4 receptors. Importantly, we found that ErbB4 is not detectable in excitatory pyramidal neurons but is abundantly expressed in GABAergic interneurons, in particular fast-spiking parvalbumin-positive basket cells. These observations strongly suggested that effects of NRG/ErbB4 on glutamatergic synaptic plasticity are indirect. To address this question directly, we compared the effects of full vs. parvalbumin (PV) interneuron-specific Erbb4 ablation in genetically engineered mice. As expected, NRG-mediated inhibition of LTP induction and reversal of early-phase LTP were absent in full ErbB4 KO mice. Interestingly, ErbB4 ablation in PV interneurons was sufficient to reproduce the effects of the full ErbB4 knockout. We also compared these two ErbB4 mutant mouse strains in a battery of behavioral tests relevant for psychiatric disorders and found that PV-restricted mutants replicated many, albeit not all, abnormalities observed in full ErbB4 mutant mice. These findings highlight the role of PV interneurons as a critical nexus of NRG/ErbB4 signaling and support a possible contribution of this pathway, genetically linked with increased risk for schizophrenia, to the pathophysiology underlying psychiatric disorders. 3. NRG/ErbB4 and dopamine D4 receptor signaling converge in PV neurons and regulate gamma oscillations: We previously reported that NRG/ErbB4 signaling modulates the power of kainate-induced hippocampal gamma oscillations. Because NRG1 dramatically increases extracellular dopamine levels in the hippocampus, we investigated the relationship between NRG/ErbB and dopamine signaling in hippocampal gamma oscillations. In collaboration with Dr.Fisahns lab, we used selective agonists for different dopamine G-coupled receptors that increase (D1 and D5 receptors) or decrease (D2, D3 and D4 receptors) the synthesis of cAMP, and found that only an agonist specific for D4 receptors (PD168077) augmented the power of gamma oscillations. By contrast, agonists for D1/D5 and D2R/D3 receptors were without effect. The increase in gamma oscillation power induced by PD168077 was totally blocked by a highly specific D4 receptor antagonist (L-745,870), further stressing the importance of this receptor for neuronal network activity. Importantly, we found that L-745,870 and clozapine, an antipsychotic that preferentially targets D4 receptors, also blocked increases in gamma oscillation power by NRG1. Using double in situ hybridization (in collaboration with Dr. U. Winzer-Serhan) and immunofluorescence histochemistry, we showed that hippocampal D4 receptor mRNA and protein are highest in a subset of GABAergic interneurons. Importantly, D4 and ErbB4 receptors are coexpressed in PV+ fast-spiking basket cells, a type of somatic-targeting GABAergic interneurons that are critically important for regulating gamma oscillations. This novel cross-talk between D4 and ErbB4 receptor signaling to augment gamma oscillation power, and their coexpression in PV-positive interneurons, suggest a cellular mechanism that may be compromised in different psychiatric disorders affecting cognitive control. 4. Neuregulin directly decreases voltage-gated sodium currents in hippocampal ErbB4-expresssing interneurons: One of our primary goals has been to investigate if and how NRG1 may directly affect the intrinsic properties of ErbB4-positive (ErbB4+) interneurons. To this end, we resorted to using dissociated hippocampal cultures, which are devoid of all hippocampal-projecting afferent connections, and to blocking all synaptic activity pharmacologically. We initially analyzed the effects of NRG1 on the excitability of ErbB4+ and ErbB4- neurons, identified in live-labeling by an ectodomain-targeting antibody, using whole-cell current-clamp recordings. We found that NRG1 decreased firing of ErbB4+ but not ErbB4- neurons by shifting the action potential threshold. Using voltage-clamp recordings, we determined that these effects are primarily attributable to decreased voltage-gated sodium channel activity, as current density was attenuated by 60% after 20 minutes of NRG1 treatment. This was the first study to identify direct actions of NRG1 on voltage-gated sodium channel function in ErbB4-expressing interneurons, thereby offering novel insights into how NRG1/ErbB4 signaling can directly impact GABAergic interneuron activity and potentially affect excitatory/inhibitory balance. 5. NRG1 signaling directly influences AMPA receptor (AMPAR) activity in glutamatergic ErbB4-expressing cerebellar granule cells (CGCs): Previous studies demonstrated how NRG1 indirectly affects plasticity at glutamatergic synapses in principal glutamatergic neurons in the hippocampus and frontal cortex. We analyzed the effects of NRG1 on developing cultured CGCs in collaboration with Dr. C. Fenster. These cultures consist predominantly of granule neurons that express ErbB4. We found that NRG1 does not affect whole-cell AMPAR or NMDAR mediated currents, nor the frequency or amplitude of spontaneous NMDAR- or AMPAR-mediated miniature excitatory post-synaptic currents, in baseline conditions of CGCs grown for 10-12 days in vitro. However, we found that high-glycine induces a form of chemical potentiation (chemLTP) in CGCs characterized by an increase in AMPAR-mEPSC frequency that is decreased by NRG1 treatment. Our data suggest that the NRG1 effect is mediated via GluA4 subunits in CGCs. This study for the first time shows that high-glycine can induce plasticity at glutamatergic synapses in CGCs, and that acute NRG1/ErbB signaling can regulate glutamatergic plasticity in CGCs in a fashion similar to SC-CA1 synapses. 6. Proteomics approach to understanding NRG/ErbB4 proximal signaling targets: To discover potentially novel and direct biological targets of NRG/ErbB4, in collaboration with Dr. S. Markeys group (NIMH), we are employing an unbiased liquid chromatography-tandem mass spectrometry (LC/MS/MS) proteomics approach to determine the composition of the ErbB4 receptor proteome. We recently reported validation of using our receptor mAb for affinity purification ErbB4 complexes and their analysis by LC/MS/MS. We are using a similar approach to isolate endogenous ErbB4 complexes from metabolically active synaptosomes following NRG-1 stimulation. Using this approach, we discovered that NRG/ErbB4 signaling alters the ion channel composition of cellular microdomains associated with ErbB4. Our goal is to determine the functional consequences of these alterations. |
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