Activity-Dependent Bidirectional Regulation of GAD Expression in a Homeostatic Fashion Is Mediated by BDNF-Dependent and Independent Pathways

RESEARCH ARTICLE Activity-Dependent Bidirectional Regulation of GAD Expression in a Homeostatic Fashion Is Mediated by BDNF-Dependent and Independent Pathways Yoko Hanno-Iijima1,2, Masami Tanaka1,2, Takatoshi Iijima1,2* a11111 1 Tokai University Institute of Innovative Science and Technology, Medical Division, Kanagawa, Japan, 2 School of Medicine, Tokai University, Kanagawa, Japan * Abstract OPEN ACCESS Citation: Hanno-Iijima Y, Tanaka M, Iijima T (2015) Activity-Dependent Bidirectional Regulation of GAD Expression in a Homeostatic Fashion Is Mediated by BDNF-Dependent and Independent Pathways. PLoS ONE 10(8): e0134296. doi:10.1371/journal. pone.0134296 Editor: Michal Hetman, University of Louisville, UNITED STATES Received: April 14, 2015 Accepted: July 7, 2015 Published: August 4, 2015 Copyright: © 2015 Hanno-Iijima et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by Innovative Academic Promotional System in Private Schools Capable of Reproducing First-Rate Researchers under Improvement of Independent Research Environment for Young Researchers Program” organized by the Ministry of Education, Culture, Sports, Science and Technology (JST) (to Tokai Univ. IIST), the Mitsubishi Foundation (Mitsubishi zaidan) (T.I.), Yamada Science Foundation (T.I.) and Takeda Science Foundation (T.I.). Homeostatic synaptic plasticity, or synaptic scaling, is a mechanism that tunes neuronal transmission to compensate for prolonged, excessive changes in neuronal activity. Both excitatory and inhibitory neurons undergo homeostatic changes based on synaptic transmission strength, which could effectively contribute to a fine-tuning of circuit activity. However, gene regulation that underlies homeostatic synaptic plasticity in GABAergic (GABA, gamma aminobutyric) neurons is still poorly understood. The present study demonstrated activity-dependent dynamic scaling in which NMDA-R (N-methyl-D-aspartic acid receptor) activity regulated the expression of GABA synthetic enzymes: glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67). Results revealed that activity-regulated BDNF (brainderived neurotrophic factor) release is necessary, but not sufficient, for activity-dependent up-scaling of these GAD isoforms. Bidirectional forms of activity-dependent GAD expression require both BDNF-dependent and BDNF-independent pathways, both triggered by NMDA-R activity. Additional results indicated that these two GAD genes differ in their responsiveness to chronic changes in neuronal activity, which could be partially caused by differential dependence on BDNF. In parallel to activity-dependent bidirectional scaling in GAD expression, the present study further observed that a chronic change in neuronal activity leads to an alteration in neurotransmitter release from GABAergic neurons in a homeostatic, bidirectional fashion. Therefore, the differential expression of GAD65 and 67 during prolonged changes in neuronal activity may be implicated in some aspects of bidirectional homeostatic plasticity within mature GABAergic presynapses. Introduction Homeostatic synaptic plasticity tunes synaptic strength to compensate for prolonged, excessive changes in neuronal activity [1]. This plasticity is also thought to play an important role in the PLOS ONE | DOI:10.1371/journal.pone.0134296 August 4, 2015 1 / 18 Activity-Dependent Bidirectional Regulation of GAD Expression Competing Interests: The authors have declared that no competing interests exist. maintenance of proper network activity. Several studies have revealed the precise mechanisms underlying activity-dependent changes in strength at glutamatergic synapses. For one, activitydependent scaling of excitatory synapses depends on postsynaptic activity [2]. Furthermore, prolonged changes in neuronal activity lead to alterations in the number of synaptic α-amino3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPA-Rs) through protein synthesis-dependent AMPA-R trafficking; this, in turn tunes excitatory synaptic transmission [3]. However, not only is excitatory synaptic strength tuned but also inhibitory synaptic strength [4] [5]. The γ-aminobutyric acid (GABA)-type inhibitory neuron regulates synaptic integration, the probability of action potential generation, and the timing of action potential generation. Impaired inhibitory functioning is one of the underlying contributory causes of neuropsychiatric disorders, as well as neuropathies characterized by brain ischemia and neuronal hyper excitability syndromes such as seizure and epilepsy [6]. Therefore, it is expected that homeostatic control of inhibitory neurons will be revealed as very important for overall network activity maintenance within neuropathological conditions. However, the gene expression changes underlying homeostatic changes at GABAergic inhibitory synapses remain unclear. One of the key factors for gene regulation during homeostatic scaling within inhibitory synapses is brain-derived neurotrophic factor (BDNF) [7] [8], which is predominantly expressed in excitatory neurons [9] [10] [11] [12]. Neuronal activity promotes BDNF expression [13] [14] [15], and a GABA-synthetic enzyme—glutamic acid decarboxylase (GAD)—is one of the major factors upregulated by long-term BDNF treatment via MAP kinase [16] [17]. GAD transcription is induced by BDNF-TrkB signaling in a RAS-ERK-CREB dependent manner [17]. Therefore, the present study’s main hypothesis was that any Ca2+-dependent dynamic GAD up-regulation is mediated by NMDA-induced BDNF that is produced and released from surrounding excitatory neurons. In fact, there is increasing evidence for a relationship between homeostatic scaling at GABAergic inhibitory synapses and BDNF-induced GAD expression. However, it remains unclear whether BDNF release is sufficient for activity-dependent GAD expression and the homeostatic functions of GABAergic inhibitory neurons. In this regard, it is relevant that GAD has two isoforms—GAD65 and GAD67—encoded by two separate genes, Gad2 and Gad1, respectively [18]; these isoforms also have different subcellular localizations and functions [18] [19] [20]. Therefore, the present study sought whether there are any differences in activity-dependent gene regulation and related neuronal functions between these two GAD isoforms. The present study revealed that activity-dependent dynamic regulation of GAD65 and GAD67 is differentially controlled by Ca2+-dependent mechanisms triggered by N-methyl-Daspartic acid receptors (NMDA-Rs). Results confirmed that activity-regulated BDNF could be a key factor in this process. However, BDNF is not sufficient for full bidirectional regulation, sugge (...truncated)