Kazuko Sakata

Critical role of promoter IV-driven BDNF transcription in GABAergic transmission and synaptic plasticity in the prefrontal cortex

Transcription of Bdnf is controlled by multiple promoters, which drive expression of multiple transcripts encoding for the same protein. Promoter IV contributes significantly to activity-dependent brain-derived neurotrophic factor (BDNF) transcription. We have generated promoter IV mutant mice (BDNF-KIV) by inserting a GFP-STOP cassette within the Bdnf exon IV locus. This genetic manipulation results in disruption of promoter IV-mediated Bdnf expression. BDNF-KIV animals exhibited significant deficits in GABAergic interneurons in the prefrontal cortex (PFC), particularly those expressing parvalbumin, a subtype implicated in executive function and schizophrenia. Moreover, disruption of promoter IV-driven Bdnf transcription impaired inhibitory but not excitatory synaptic transmission recorded from layer V pyramidal neurons in the PFC. The attenuation of GABAergic inputs resulted in an aberrant appearance of spike-timing-dependent synaptic potentiation (STDP) in PFC slices derived from BDNF-KIV, but not wild-type littermates. These results demonstrate the importance of promoter IV-dependent Bdnf transcription in GABAergic function and reveal an unexpected regulation of STDP in the PFC by BDNF.

Cloning of a lymphatic peptide/histidine transporter.

Although peptide transport across the plasma membrane has been characterized well in the kidney and the intestine, the functional relevance of this transport in other organs has not been addressed. Here we report the cloning of a cDNA for a novel peptide/histidine transporter found in the rat (rPHT2), whose mRNA is expressed mainly in the lymphatic system. rPHT2 encodes a protein of 582 amino acids and showed 49% identity with the brain PHT (PHT1) [Yamashita, Shimada, Guo, Sato, Kohmura, Hayakawa, Takagi and Tohyama (1997) J. Biol. Chem. 272, 10205-10211]. rPHT2 mRNA was abundant in lung, spleen and thymus, and detected faintly in brain, liver, adrenal gland and heart by Northern-blot analysis and reverse transcriptase PCR (RT-PCR). Intense signals for the gene were found in immunocytes using in situ hybridization. Ectopic expression of rPHT2 protein in HEK-293T cells and BHK cells was not found on the cell surface, but was found on the lysosomal membrane using light- and electron-microscopic analysis. Recombinant rPHT2 protein reconstituted into liposomes showed proton-dependent transport activity with histidine and histidyl-leucine. These findings suggest that rPHT2 is involved in the protein catabolic pathway in the lymphatic system.

Altered Synapse Formation in the Adult Somatosensory Cortex of Brain-Derived Neurotrophic Factor Heterozygote Mice

Increased sensory stimulation in the adult whisker-to-barrel pathway induces the expression of BDNF as well as synapse formation in cortical layer IV. Here, we investigated whether BDNF plays a role in the alterations of connectivity between neurons by analyzing the ultrastructure of the BDNF heterozygote mouse, characterized by a reduced level of BDNF expression. Using serial section electron microscopy, we measured synapse density, spine morphology, and synaptic vesicle distribution to show that mice with a reduced level of BDNF have a barrel neuropil that is indistinguishable from wild-type controls. After 24 hr of whisker stimulation, however, there is no indication of synapse formation in the heterozygous mouse. Whereas the balance between excitatory and inhibitory synapses is modified in the controls, it remains constant in the heterozygotes. The distribution of synaptic vesicles in excitatory synapses is the same in heterozygous and wild-type mice and is not influenced by the stimulation paradigm. Spine volume, however, is unchanged by stimulation in the wild-type animals, but does increase significantly in the heterozygous animal. These results provide evidence that, in vivo, BDNF plays an important role in the structural rearrangement of adult cortical circuitry as a consequence of an increased sensory input.

Role of activity-dependent BDNF expression in hippocampal–prefrontal cortical regulation of behavioral perseverance

Significance The brain-derived neurotrophic factor (Bdnf) gene is expressed either constitutively or in an activity-dependent manner. Selective disruption of activity-dependent brain-derived neurotrophic factor protein expression (BDNF-KIV mice) results in specific deficits in GABAergic transmission in the prefrontal cortex (PFC) and a form of long-term synaptic plasticity in the hippocampus. Surprisingly, PFC-dependent working memory and hippocampus-dependent spatial and fear memory are normal. In contrast, BDNF-KIV mice exhibit pronounced behavioral perseverance, as reflected by impairments in spatial memory reversal and fear memory extinction. Thus, activity-dependent BDNF expression in hippocampal–PFC circuits may play an important role in cognitive behavior in vivo. Our study also may shed light on the pathogenesis of several cognitive disorders in which perseverance is prominent, including schizophrenia and posttraumatic stress disorder. Activity-dependent gene transcription, including that of the brain-derived neurotrophic factor (Bdnf) gene, has been implicated in various cognitive functions. We previously demonstrated that mutant mice with selective disruption of activity-dependent BDNF expression (BDNF-KIV mice) exhibit deficits in GABA-mediated inhibition in the prefrontal cortex (PFC). Here, we show that disruption of activity-dependent BDNF expression impairs BDNF-dependent late-phase long-term potentiation (L-LTP) in CA1, a site of hippocampal output to the PFC. Interestingly, early-phase LTP and conventional L-LTP induced by strong tetanic stimulation were completely normal in BDNF-KIV mice. In parallel, attenuation of activity-dependent BDNF expression significantly impairs spatial memory reversal and contextual memory extinction, two executive functions that require intact hippocampal–PFC circuitry. In contrast, spatial and contextual memory per se were not affected. Thus, activity-dependent BDNF expression in the hippocampus and PFC may contribute to cognitive and behavioral flexibility. These results suggest distinct roles for different forms of L-LTP and provide a link between activity-dependent BDNF expression and behavioral perseverance, a hallmark of several psychiatric disorders.

Expression pattern of messenger RNAs for prostanoid receptors in glial cell cultures

Expression level of messenger RNAs (mRNAs) for prostanoid EP3, FP, and TP receptors was investigated in cultured rat astrocytes, oligodendrocytes, and microglia, as well as in meningeal fibroblasts, rat glioma C6 cells, rat pheochromocytoma PC12 cells, whole brain, and several peripheral tissues by reverse transcriptase-polymerase chain reaction. Cultured astrocytes and oligodendrocytes expressed mRNAs for 3 prostanoid receptors examined. In contrast, cultured microglia and pheochromocytoma PC12 cells expressed EP3 and TP receptor mRNAs, but not FP receptor mRNA. Glioma C6 cells expressed only TP receptor mRNA among 3 prostanoid receptors with the same expression level as that in astrocytes. Cultured meningeal fibroblasts expressed 3 receptor transcripts, and their expression levels were lower than those in astrocytes. Expression level of mRNA for each prostanoid receptor in cultured glial cells was higher than that in whole brain. These observations suggest that each prostanoid has its specific roles in each glial cell type of the brain.

A key mechanism underlying sensory experience-dependent maturation of neocortical GABAergic circuits in vivo

Mechanisms underlying experience-dependent refinement of cortical connections, especially GABAergic inhibitory circuits, are unknown. By using a line of mutant mice that lack activity-dependent BDNF expression (bdnf-KIV), we show that experience regulation of cortical GABAergic network is mediated by activity-driven BDNF expression. Levels of endogenous BDNF protein in the barrel cortex are strongly regulated by sensory inputs from whiskers. There is a severe alteration of excitation and inhibition balance in the barrel cortex of bdnf-KIV mice as a result of reduced inhibitory but not excitatory conductance. Within the inhibitory circuits, the mutant barrel cortex exhibits significantly reduced levels of GABA release only from the parvalbumin-expressing fast-spiking (FS) interneurons, but not other interneuron subtypes. Postnatal deprivation of sensory inputs markedly decreased perisomatic inhibition selectively from FS cells in wild-type but not bdnf-KIV mice. These results suggest that postnatal experience, through activity-driven BDNF expression, controls cortical development by regulating FS cell-mediated perisomatic inhibition in vivo.

Lack of promoter IV-driven BDNF transcription results in depression-like behavior

Transcription of Bdnf is controlled by multiple promoters, in which promoter IV contributes significantly to activity‐dependent Bdnf transcription. We have generated promoter IV mutant mice [brain‐derived neurotrophic factor (BDNF)‐KIV] in which promoter IV‐driven expression of BDNF is selectively disrupted by inserting a green fluorescent protein (GFP)‐STOP cassette within the Bdnf exon IV locus. BDNF‐KIV animals exhibited depression‐like behavior as shown by the tail suspension test (TST), sucrose preference test (SPT) and learned helplessness test (LHT). In addition, BDNF‐KIV mice showed reduced activity in the open field test (OFT) and reduced food intake in the novelty‐suppressed feeding test (NSFT). The mutant mice did not display anxiety‐like behavior in the light and dark box test and elevated plus maze tests. Interestingly, the mutant mice showed defective response inhibition in the passive avoidance test (PAT) even though their learning ability was intact when measured with the active avoidance test (AAT). These results suggest that promoter IV‐dependent BDNF expression plays a critical role in the control of mood‐related behaviors. This is the first study that directly addressed the effects of endogenous promoter‐driven expression of BDNF in depression‐like behavior.

Hsp90 Chaperone Inhibitor 17-AAG Attenuates Aβ-Induced Synaptic Toxicity and Memory Impairment

The excessive accumulation of soluble amyloid peptides (Aβ) plays a crucial role in the pathogenesis of Alzheimers disease (AD), particularly in synaptic dysfunction. The role of the two major chaperone proteins, Hsp70 and Hsp90, in clearing misfolded protein aggregates has been established. Despite their abundant presence in synapses, the role of these chaperones in synapses remains elusive. Here, we report that Hsp90 inhibition by 17-AAG elicited not only a heat shock-like response but also upregulated presynaptic and postsynaptic proteins, such as synapsin I, synaptophysin, and PSD95 in neurons. 17-AAG treatment enhanced high-frequency stimulation-evoked LTP and protected neurons from synaptic damage induced by soluble Aβ. In AD transgenic mice, the daily administration of 17-AAG over 7 d resulted in a marked increase in PSD95 expression in hippocampi. 17-AAG treatments in wild-type C57BL/6 mice challenged by soluble Aβ significantly improved contextual fear memory. Further, we demonstrate that 17-AAG activated synaptic protein expression via transcriptional mechanisms through the heat shock transcription factor HSF1. Together, our findings identify a novel function of Hsp90 inhibition in regulating synaptic plasticity, in addition to the known neuroprotective effects of the chaperones against Aβ and tau toxicity, thus further supporting the potential of Hsp90 inhibitors in treating neurodegenerative diseases.

Enriched environment treatment reverses depression-like behavior and restores reduced hippocampal neurogenesis and protein levels of brain-derived neurotrophic factor in mice lacking its expression through promoter IV

Promoter IV-driven expression of brain-derived neurotrophic factor (BDNF), a major neuronal growth factor, is implicated in the pathophysiology of major depression. We previously reported that mice lacking expression of BDNF through promoter IV (BDNF-KIV mice) exhibit a depression-like phenotype. Here, we examined whether the depression-like phenotype and decreased levels of BDNF because of promoter IV deficit could be rescued by enriched environment (EE) treatment, a potential antidepressant intervention. Three weeks of EE treatment rescued depression-like behavior of BDNF-KIV mice as assessed by the tail suspension test, open-field test and sucrose preference test. EE treatment also increased BDNF transcripts driven by multiple endogenous promoters and restored BDNF protein levels in the hippocampus (HIP) of BDNF-KIV mice. Further, we investigated adult hippocampal neurogenesis as a possible cellular mechanism underlying the depression-like behavior and its recovery in BDNF-KIV mice. We found that the number of surviving progenitors and their dendritic length in the dentate gyrus of the HIP were reduced in BDNF-KIV mice compared with the control wild-type mice. EE treatment restored the reduction in cell survival and dendritic length and increased cell proliferation in BDNF-KIV mice. In conclusion, this study demonstrated that EE rescued depression-like behavior, decreased BDNF levels and defective neurogenesis in the HIP caused by lack of promoter IV-driven BDNF expression. These results suggest that decreased BDNF levels because of one impaired promoter can be compensated by other BDNF promoters and that BDNF levels may be one of the key factors regulating depression and antidepressant effects through hippocampal neurogenesis.

Characterization of glycine release mediated by glycine transporter 1 stably expressed in HEK-293 cells

We constructed a cell line which stably expresses glycine transporter 1 (GlyT1) proteins. The cell line showed significant [14C]glycine uptake and could keep steep glycine concentration gradient between intracellular and extracellular space (in > out). Using this cell line, we investigated glycine release mediated by this transporter. The [14C]glycine release was enhanced by extracellular glycine and sarcosine, a selective inhibitor of the transporter, in a dose-dependent manner. In addition, the replacement of extracellular Na+ with Li+ or extracellular Cl- with acetate- markedly increased the release. Furthermore, we investigated the effects of extracellular Ca2+ and K+. The removal of these ions also showed enhancement of the release. These results suggest that glycine transporter 1 protein, which might be involved in the NMDA receptor neurotransmission, can release glycine into the extracellular space in the vicinity of synapses, and that the release might be influenced by the extracellular substrate concentration and ion composition in the synaptic cleft.