PSD95 and nNOS interaction as a novel molecular target to modulate conditioned fear: relevance to PTSD

Translational Psychiatry, Aug 2018

Stimulation of N-methyl-D-aspartic acid receptors (NMDARs) and the resulting increase of nitric oxide (NO) production are critical for fear memory formation. Following NMDAR activation, efficient production of NO requires linking the 95 kDa postsynaptic density protein (PSD95), a scaffolding protein to neuronal nitric oxide synthase (nNOS). A variety of previously studied NMDAR antagonists and NOS inhibitors can disrupt fear conditioning, but they also affect many other CNS functions such as motor activity, anxiety, and learning. We hypothesized that disrupting nNOS and PSD95 interaction in the amygdala, a critical site for fear memory formation, will reduce conditioned fear. Our results show that systemic treatment with ZL006, a compound that disrupts PSD95/nNOS binding, attenuates fear memory compared to its inactive isomer ZL007. Co-immunoprecipitation after fear conditioning showed a robust increase in the amygdala PSD95/nNOS binding, which was blocked by systemic pre-administration of ZL006. Treatment of amygdala slices with ZL006 also impaired long-term potentiation (LTP), a cellular signature of synaptic plasticity. Direct intra-amygdala infusion of ZL006 also attenuated conditioned fear. Finally, unlike NMDAR antagonist MK-801, ZL006 does not affect locomotion, social interaction, object recognition memory, and spatial memory. These findings support the hypothesis that disrupting the PSD95/nNOS interaction downstream of NMDARs selectively reduces fear memory, and highlights PSD95/nNOS interaction as a novel target for fear-related disorders, such as posttraumatic stress disorder.

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PSD95 and nNOS interaction as a novel molecular target to modulate conditioned fear: relevance to PTSD

Abstract Stimulation of N-methyl-D-aspartic acid receptors (NMDARs) and the resulting increase of nitric oxide (NO) production are critical for fear memory formation. Following NMDAR activation, efficient production of NO requires linking the 95 kDa postsynaptic density protein (PSD95), a scaffolding protein to neuronal nitric oxide synthase (nNOS). A variety of previously studied NMDAR antagonists and NOS inhibitors can disrupt fear conditioning, but they also affect many other CNS functions such as motor activity, anxiety, and learning. We hypothesized that disrupting nNOS and PSD95 interaction in the amygdala, a critical site for fear memory formation, will reduce conditioned fear. Our results show that systemic treatment with ZL006, a compound that disrupts PSD95/nNOS binding, attenuates fear memory compared to its inactive isomer ZL007. Co-immunoprecipitation after fear conditioning showed a robust increase in the amygdala PSD95/nNOS binding, which was blocked by systemic pre-administration of ZL006. Treatment of amygdala slices with ZL006 also impaired long-term potentiation (LTP), a cellular signature of synaptic plasticity. Direct intra-amygdala infusion of ZL006 also attenuated conditioned fear. Finally, unlike NMDAR antagonist MK-801, ZL006 does not affect locomotion, social interaction, object recognition memory, and spatial memory. These findings support the hypothesis that disrupting the PSD95/nNOS interaction downstream of NMDARs selectively reduces fear memory, and highlights PSD95/nNOS interaction as a novel target for fear-related disorders, such as posttraumatic stress disorder. Introduction Normal fear learning and memory allow animals to predict and avoid physical dangers and are therefore essential to survival. However, following traumatic experiences, these mechanisms can lead to symptoms of syndromes such as posttraumatic stress disorder (PTSD)1,2. PTSD is a severe psychiatric disorder in which fear responses are likely sustained, generalized, and inappropriately triggered out of context1,2. Pavlovian fear conditioning is a well-established laboratory model of fear learning that is often used to elucidate mechanism of fear acquisition and extinction. In this paradigm, a neutral event (a conditioned stimulus, (CS)), such as a tone, is paired with an aversive event (an unconditioned stimulus (US)), such as a footshock. Once learned, the CS acquires the ability to evoke fear responses, such as freezing in anticipation of the US3. Pavlovian fear conditioning is known to be dependent on the synaptic plasticity within the amygdala4,5 and is mediated by excitatory neurotransmission acting through N-methyl-D-aspartic acid receptors (NMDARs). A number of studies have demonstrated a critical role of NMDARs in fear conditioning. For example, systemic and CNS site-specific administration of NMDAR antagonists block fear acquisition when given before training6,7,8,9 and impair fear expression when administered before fear recall10,11. Unfortunately, despite this important role for NMDARs in impairing fear formation, NMDAR antagonists have limited therapeutic potential due to their significant adverse side-effect profiles12,13. Stimulation of NMDARs activates a number of downstream signaling pathways. One such downstream effect involves activation of the enzyme neuronal nitric oxide synthase (nNOS) and the resulting production of the signaling molecule nitric oxide (NO). nNOS is one of three isoforms of NOS (the other isoforms being endothelial NOS and inducible NOS) and is preferentially expressed in neurons and functionally coupled to NMDAR signaling14. There is strong evidence that activation of nNOS following NMDAR activation is a critical component of fear memory formation15,16. Indeed, pharmacological inhibition of enzyme activity and gene deletion of nNOS have been shown to reduce fear. For example, systemic and intra-amygdala administration of NOS inhibitors reduce fear memories in multiple models of fear conditioning17,18; mice with nNOS gene knockout display impairments in both contextual and cued fear learning19. Despite being downstream of NMDARs, unfortunately, global inhibition of nNOS enzyme itself cause undesired systemic effects, such as deficits in motor functions20,21,22 and impairments in some other forms of learning23,24,25,26. Therefore, similar to direct NMDAR antagonism, therapeutic targeting of the downstream nNOS enzyme is undesirable due to adverse effects. Following NMDAR activation, nNOS binds to the scaffolding protein postsynaptic density protein 95 kDa (PSD95), and this is a required step for the efficient production of NO14. Thus, selective disruption of the PSD95/nNOS binding would allow a targeted approach to specific reduction of NO production during high glutamate neurotransmission state without affecting normal intracellular nNOS functions. By not disrupting NMDAR-dependent signaling pathways, this approach could circumvent the adverse effects associat (...truncated)


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L.- P. Li, E. T. Dustrude, M. M. Haulcomb, A. R. Abreu, S. D. Fitz, P. L. Johnson, G. A. Thakur, A. I. Molosh, Y. Lai, A. Shekhar. PSD95 and nNOS interaction as a novel molecular target to modulate conditioned fear: relevance to PTSD, Translational Psychiatry, 2018, Issue: 8, DOI: 10.1038/s41398-018-0208-5