Working memory deficits in neuronal nitric oxide synthase knockout mice: Potential impairments in prefrontal cortex mediated cognitive function

Abstract

Neuronal nitric oxide synthase (nNOS) forms nitric oxide (NO), which functions as a signaling molecule via S-nitrosylation of various proteins and regulation of soluble guanylate cyclase (cGC)/cyclic guanosine monophosphate (cGMP) pathway in the central nervous system. nNOS signaling regulates diverse cellular processes during brain development and molecular mechanisms required for higher brain function. Human genetics have identified nNOS and several downstream effectors of nNOS as risk genes for schizophrenia. Besides the disease itself, nNOS has also been associated with prefrontal cortical functioning, including cognition, of which disturbances are a core feature of schizophrenia. Although mice with genetic deletion of nNOS display various behavioral deficits, no studies have investigated prefrontal cortex-associated behaviors. Here, we report that nNOS knockout (KO) mice exhibit hyperactivity and impairments in contextual fear conditioning, results consistent with previous reports. nNOS KO mice also display mild impairments in object recognition memory. Most importantly, we report for the first time working memory deficits, potential impairments in prefrontal cortex mediated cognitive function in nNOS KO mice. Furthermore, we demonstrate Disrupted-in-Schizophrenia 1 (DISC1), another genetic risk factor for schizophrenia that plays roles for cortical development and prefrontal cortex functioning, including working memory, is a novel protein binding partner of nNOS in the developing cerebral cortex. Of note, genetic deletion of nNOS appears to increase the binding of DISC1 to NDEL1, regulating neurite outgrowth as previously reported. These results suggest that nNOS KO mice are useful tools in studying the role of nNOS signaling in cortical development and prefrontal cortical functioning.

Introduction

Cognition is an important higher brain function that controls behavioral outcomes. Many brain regions, including the prefrontal cortex, have been implicated in mediating key cognitive processes. Disturbances in such areas ultimately result in a wide range of cognitive deficits, which have been frequently reported as core features of neuropsychiatric disorders, such as schizophrenia. Thus, understanding cognitive function at the molecular, circuit, and behavioral levels becomes extremely valuable when studying pathophysiological mechanisms of neuropsychiatric conditions.

The association of genetic risk factors for major mental disorders and cognition has been widely examined in human studies [1], [2]. For example, neuronal nitric oxide synthase (nNOS), a risk gene for schizophrenia [3], [4], has been associated with prefrontal cortical functioning, including cognition, as assessed by neurospsychological testing in patients with schizophrenia as well as healthy subjects [4], [5]. These results suggest the involvement of nNOS not only in the etiopathogenesis of schizophrenia, but with specific cognitive phenotypic domains. Additionally, many rare structural variants in multiple genes in NOS signaling are disrupted in patients with schizophrenia [6]. Several downstream effectors in nNOS signaling, such as CAPON and serine racemase [7], [8], have also been reportedly associated with schizophrenia [9], [10].

NO, a gaseous neurotransmitter produced by nNOS, is an essential messenger in diverse developmental processes involved in neural circuit formation, ranging from the refinement of axonal projections to the regulation of dendrite and spine morphologies [11], [12]. Consistent with its roles during brain development, nNOS is highly expressed in the developing cerebral cortex, whereas its expression is diminished to mainly a subset of interneurons at adult stages [13]. A major mechanism of action of NO is S-nitrosylation on cysteine residues of target proteins, resulting in significant conformational changes that affect their functional activity [14]. Another critical route for NO-mediated neuronal processes is the activation of soluble guanylate cyclase (sGC) to catalyze the production of cyclic guanosine monophosphate (cGMP), a second messenger for many cellular processes, including axon/dendrite growth and synaptogenesis [15], [16]. Several phosphodiesterases (PDE), enzymes that regulate the levels of cGMP, have been genetically associated with schizophrenia [17]. Nonetheless, even though such a variety of nNOS related functions have been studied at the molecular levels, their impact on behaviors remains to be elucidated.

In this regard, genetic deletion of nNOS in animal models is a useful tool in studying the role of nNOS in brain function and behavior [18]. nNOS knockout (KO) mice have been characterized by an increase in aggressive behavior and sexual behavior, as well as hyperactivity and abnormal social behavior [19], [20]. No consistent results have been reported in anxiety-related behaviors as some studies suggest increased anxiety levels [21], while others stress anxiolytic-like phenotypes [22]. Contextual fear conditioning has been reported to be significantly impaired [23]. Impairments in cognitive functions, such as spatial reference and spatial working memory, have been reported [20], [21], [24]. Nonetheless, cognitive functions dependent upon the integrity of prefrontal cortical regions remain to be studied.

In this study, we characterized cognition, including prefrontal cortex mediated behaviors in nNOS KO mice. Most importantly, we found working memory deficits in nNOS KO mice. Furthermore, we demonstrated Disrupted-in-Schizophrenia 1 (DISC1), another genetic risk factor for schizophrenia that plays roles for cortical development and prefrontal cortex functioning [25], [26], [27], including working memory, is a novel protein binding partner of nNOS in the developing cerebral cortex. Moreover, we found that nNOS influences the interaction of DISC1 and nuclear distribution element-like 1 (NDEL1), interaction which regulates neurite outgrowth as previously reported [28], [29]. Our findings implicate nNOS signaling in cortical development and potential prefrontal cortical functioning.