This year we have made a significant progress in revealing several new aspects of neurotrophin functions. ? ? 1) Distinct Role of Long 3UTR BDNF mRNA in Spine Morphology and Synaptic Plasticity in Hippocampal Pyramidal Neurons ? A quite common and yet puzzling phenomenon in cell biology is the existence of transcripts that code for exactly the same protein but with different 3 untranslated region (3UTR). One such example is the messenger RNA for BDNF. There are two pools of BDNF mRNAs in neurons with similar abundance: one with a short 3UTR) and the other with a long 3UTR. It is not known why a neuron would need two mRNAs of different length, coding for the same BDNF protein. We have now provided evidence suggesting that mRNAs of different 3UTR confer differential functions due to their distinct subcellular localization. We show that the short 3UTR BDNF mRNAs are restricted in neuronal cell bodies whereas the long 3UTR BDNF mRNAs are also localized in dendrites. In a mouse mutant where the long 3UTR is truncated, dendritic targeting of BDNF mRNAs is impaired. There is little BDNF in hippocampal dendrites despite normal levels of total BDNF protein. This mutant exhibits deficits in pruning and enlargement of dendritic spines, as well as selective impairment in long-term potentiation in dendrites, but not somata, of hippocampal neurons. These results are significant for several reasons. First, it elucidates a cellular mechanism why BDNF, a key regulator for brain development and plasticity, could elicit diverse cellular functions ranging from cell survival to synaptic transmission. Second, it provides clear experimental evidence that dendritically localized mRNAs play crucial roles in regulating spine morphologies, a long-sought result in the field of local protein synthesis and synaptic plasticity. Finally, as far as we know, this is the first demonstration that different transcripts with the same coding sequence have distinct roles in a cell.? ? 2) Activity-dependent BDNF transcription and secretion? In the first study, we address the role of activity-dependent BDNF gene expression. Transcription of the BDNF gene is controlled by multiple promoters, which drive the expression of multiple transcripts that encode for the same protein. Promoter-IV contributes significantly to activity-dependent BDNF transcription. We have generated promoter-IV mutant mice (BDNF-KIV) by inserting the GFP gene followed by a stop codon into exon-IV. This genetic manipulation resulted in disruption of promoter-IV-mediated BDNF expression, particularly in the prefrontal cortex (PFC), an area involved in working memory and other executive functions. Interestingly, the BDNF-KIV exhibited a selective impairment in parvalbumin-positive GABAergic neurons and inhibitory but not excitatory postsynaptic currents in the PFC, leading to an aberrant spike-timing dependent synaptic potentiation (STDP). Behaviorally, BDNF-KIV mice are significantly impaired in PFC-mediated reversal learning and fear memory extinction, but not working memory. These results demonstrate the importance of promoter-IV-dependent BDNF transcription in GABAergic function, and reveal an unexpected role of BDNF in behavioral perseverance. Our study may shed light on the pathogenesis of several cognitive disorders in which perseverance is prominent, including schizophrenia and Post-traumatic stress disorder (PTSD).? In the second study, we address the role of activity-dependent BDNF gene expression. We have previously shown that pro- and mature BDNF often elicit opposing biological effects. For instance, mature BDNF (mBDNF) is critical for long-term potentiation (LTP) induced by high-frequency stimulation (HFS), whereas proBDNF facilitate long-term depression (LTD) induced by low-frequency stimulation (LFS). Since mBDNF is derived from proBDNF by endoproteolytic cleavage, mechanisms regulating the cleavage of proBDNF may control the direction of BDNF regulation. Using methods that selectively detect proBDNF or mBDNF, we show that LFS induced predominant proBDNF secretion in cultured hippocampal neurons. In contrast, HFS preferentially increased extracellular mBDNF. Inhibition of extracellular, but not intracellular cleavage of proBDNF greatly reduced HFS-induced extracellular mBDNF. Moreover, HFS, but not LFS, selectively induced the secretion of tPA, a key protease involved in extracellular proBDNF to mBDNF conversion. Thus, high-frequency neuronal activity controls the ratio of extracellular proBDNF/mBDNF by regulating the secretion of extracellular proteases. Our study demonstrates activity-dependent control of extracellular proteolytic cleavage of a secretory protein, and reveals an important mechanism that controls diametrically opposed functions of BDNF isoforms. In addition to revealing the first example of how neuronal activity can control the cleavage of a secreted protein, this work provides useful tools to study distinct function of proBDNF and mBDNF.? ? 3) Studies of genes involved in schizophrenia? An important goal of modern medicine is to understand the basic mechanisms underlying common and complex human diseases, such as diabetes and schizophrenia. Conventional genetic linkage and association studies have confirmed that these diseases involve multiple genes, and each gene elicits very small effects across populations. Moreover, because of the complexity of genetic factors and their interaction with the environment, it is often difficult to link genetic findings to important biological or mechanistic aspects of the illness. To overcome these obstacles, we have taken a powerful translational approach that dissects the complex phenomenology of psychosis into several neural system and molecular components and maps genetic association onto these multiple levels of analysis. Using this approach, we identify a potentially new class of schizophrenia susceptibility genes, the KCNH2 potassium channel, but more importantly, a novel isoform of this type of potassium channel that is primate and brain specific and increased 2.5 fold in schizophrenia brain tissue. Expression of this novel isoform is predicted by risk associated SNPs in the gene, even in normal brain tissue, and risk SNPs also predict cognition and related brain physiology in normal subjects. Postmortem expression analysis shows a 2.5-fold increase in Isoform 3.1 relative to KCNH2-1A in schizophrenic hippocampus. Structurally, Isoform 3.1 lacks most of the PAS domain critical for slow channel deactivation. Electrophysiological characterization in primary cortical neurons reveals that overexpression of Isoform 3.1 results in a rapidly deactivating K+ current and a high-frequency, non-adapting firing pattern. These results identify a novel KCNH2 channel involved in cortical physiology, cognition, and psychosis, providing a potential new target for psychotherapeutic drugs.
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