Decreased nesting behavior, selective increases in locomotor activity in a novel environment, and paradoxically increased open arm exploration in Neurogranin knockout mice.

AIMS: Neurogranin (NRGN) is a postsynaptic protein kinase substrate that binds calmodulin in the absence of calcium. Recent studies suggest that NRGN is involved in neuropsychiatric disorders, including schizophrenia, ADHD, and Alzheimer's disease. Previous behavioral studies of Nrgn knockout (Nrgn KO) mice identified hyperactivity, deficits in spatial learning, impaired sociability, and decreased prepulse inhibition, which suggest that these mice recapitulate some symptoms of neuropsychiatric disorders. To further validate Nrgn KO mice as a model of neuropsychiatric disorders, we assessed multiple domains of behavioral phenotypes in Nrgn KO mice using a comprehensive behavioral test battery including tests of homecage locomotor activity and nesting behavior. METHODS: Adult Nrgn KO mice (28-54 weeks old) were subjected to a battery of comprehensive behavioral tests, which examined general health, nesting behavior, neurological characteristics, motor function, pain sensitivity, locomotor activity, anxiety-like behavior, social behavior, sensorimotor gating, depression-like behavior, and working memory. RESULTS: The Nrgn KO mice displayed a pronounced decrease in nesting behavior, impaired motor function, and elevated pain sensitivity. While the Nrgn KO mice showed increased locomotor activity in the open field test, these mice did not show hyperactivity in a familiar environment as measured in the homecage locomotor activity test. The Nrgn KO mice exhibited a decreased number of transitions in the light-dark transition test and decreased stay time in the center of the open field test, which is consistent with previous reports of increased anxiety-like behavior. Interestingly, however, these mice stayed on open arms significantly longer than wild-type mice in the elevated plus maze. Consistent with previous studies, the mutant mice exhibited decreased prepulse inhibition, impaired working memory, and decreased sociability. CONCLUSIONS: In the current study, we identified behavioral phenotypes of Nrgn KO mice that mimic some of the typical symptoms of neuropsychiatric diseases, including impaired executive function, motor dysfunction, and altered anxiety. Most behavioral phenotypes that had been previously identified, such as hyperlocomotor activity, impaired sociability, tendency for working memory deficiency, and altered sensorimotor gating, were reproduced in the present study. Collectively, the behavioral phenotypes of Nrgn KO mice detected in the present study indicate that Nrgn KO mice are a valuable animal model that recapitulates a variety of symptoms of neuropsychiatric disorders, such as schizophrenia, ADHD, and Alzheimer's disease.

Neurogranin and VILIP-1 as Molecular Indicators of Neurodegeneration in Alzheimer's Disease: A Systematic Review and Meta-Analysis.

Neurogranin (Ng) and visinin-like protein 1 (VILIP-1) are promising candidates for Alzheimer's Disease (AD) biomarkers closely related to synaptic and neuronal degeneration. Both proteins are involved in calcium-mediated pathways. The meta-analysis was performed in random effects based on the ratio of means (RoM) with calculated pooled effect size. The diagnostic utility of these proteins was examined in cerebrospinal fluid (CSF) of patients in different stages of AD compared to control (CTRL). Ng concentration was also checked in various groups with positive (+) and negative (-) amyloid beta (Aß). Ng highest levels of RoM were observed in the AD (n = 1894) compared to CTRL (n = 2051) group (RoM: 1.62). Similarly, the VILIP-1 highest values of RoM were detected in the AD (n = 706) compared to CTRL (n = 862) group (RoM: 1.34). Concentrations of both proteins increased in more advanced stages of AD. However, Ng seems to be an earlier biomarker for the assessment of cognitive impairment. Ng appears to be related with amyloid beta, and the highest levels of Ng in CSF was observed in the group with pathological Aß+ status. Our meta-analysis confirms that Ng and VILIP-1 can be useful CSF biomarkers in differential diagnosis and monitoring progression of cognitive decline. Although, an additional advantage of the protein concentration Ng is the possibility of using it to predict the risk of developing cognitive impairment in normal controls with pathological levels of Aß1-42. Analyses in larger cohorts are needed, particularly concerning Aß status.

Neurogranin stimulates Ca2+/calmodulin-dependent kinase II by suppressing calcineurin activity at specific calcium spike frequencies.

Calmodulin sits at the center of molecular mechanisms underlying learning and memory. Its complex and sometimes opposite influences, mediated via the binding to various proteins, are yet to be fully understood. Calcium/calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN) both bind open calmodulin, favoring Long-Term Potentiation (LTP) or Depression (LTD) respectively. Neurogranin binds to the closed conformation of calmodulin and its impact on synaptic plasticity is less clear. We set up a mechanistic computational model based on allosteric principles to simulate calmodulin state transitions and its interactions with calcium ions and the three binding partners mentioned above. We simulated calcium spikes at various frequencies and show that neurogranin regulates synaptic plasticity along three modalities. At low spike frequencies, neurogranin inhibits the onset of LTD by limiting CaN activation. At intermediate frequencies, neurogranin facilitates LTD, but limits LTP by precluding binding of CaMKII with calmodulin. Finally, at high spike frequencies, neurogranin promotes LTP by enhancing CaMKII autophosphorylation. While neurogranin might act as a calmodulin buffer, it does not significantly preclude the calmodulin opening by calcium. On the contrary, neurogranin synchronizes the opening of calmodulin's two lobes and promotes their activation at specific frequencies. Neurogranin suppresses basal CaN activity, thus increasing the chance of CaMKII trans-autophosphorylation at high-frequency calcium spikes. Taken together, our study reveals dynamic regulatory roles played by neurogranin on synaptic plasticity, which provide mechanistic explanations for opposing experimental findings.

Intrahippocampal injection of a lentiviral vector expressing neurogranin enhances cognitive function in 5XFAD mice.

Progressive cognitive declines are the main clinical symptoms of Alzheimer's disease (AD). Cognitive impairment in AD is directly correlated with amyloid beta (Aß)-mediated synaptic deficits. It is known that upregulation of neurogranin (Ng), a postsynaptic protein, contributes to the enhancement of synaptic plasticity and cognitive function. By contrast, downregulation of Ng expression results in learning and memory impairments. Interestingly, Ng expression is significantly reduced in the parenchyma of brains with AD. However, the pathological role that downregulated Ng plays in the cognitive dysfunctions observed in AD remains unclear. Therefore, the present study examined whether enhancing Ng expression affected cognitive functions in 5XFAD mice, an animal model of AD. We found that the Ng reductions and cognitive decline observed in 5XFAD mice were restored in mice that were intrahippocampally injected with an Ng-expressing lentiviral vector. Furthermore, overexpression of Ng upregulated expression of postsynaptic density protein-95 in the hippocampus of 5XFAD mice. These results suggest that the cause of cognitive decline in AD may be at least partially associated with reduced Ng levels, and thus, supplementation of Ng may be an appropriate therapeutic strategy for individuals with AD.

RC3/neurogranin negatively regulates extracellular signal-regulated kinase pathway through its interaction with Ras.

RC3/neurogranin is a postsynaptic protein and plays pivotal roles in spatial learning and emotional anxiety as well as synaptic plasticity. The expression level of RC3 is dynamically changed during developmental stages, but the function of RC3 in brain development is not well understood yet. Neurotrophins interact with tropomyosin-related kinase receptors to activate Ras-extracellular signal-regulated kinase (ERK) pathway and can also induce neuronal differentiation. In this study, we demonstrate that RC3 inhibits Ras-ERK pathway by interaction with Ras and controls neurite outgrowth induced by neurotrophins. In PC12 cells, RC3 inhibits nerve growth factor (NGF)-induced activation of Ras and thereby ERK1/2 signaling cascade as well as neurite outgrowth induced by NGF. We found Ras is the target of the inhibitory function of RC3, because RC3 interacts with Ras and suppresses the elevated affinity of Ras to Ras-binding domain of Raf-1. Meanwhile, already activated Raf-1 by Ras activity is not affected by RC3. Furthermore, depletion of RC3 by RNA interference drastically enhances the stimulation of ERK1/2 and neurite outgrowth induced by brain-derived neurotrophic factor in hippocampal neurons. These findings suggest that RC3 is a novel natural inhibitor of Ras-ERK1/2 signaling axis, leading to negatively regulate neuronal differentiation induced by neurotrophins.

The effect of neurogranin on neural correlates of episodic memory encoding and retrieval.

Neurogranin (NRGN) is the main postsynaptic protein regulating the availability of calmodulin-Ca(2+) in neurons. NRGN is expressed exclusively in the brain, particularly in dendritic spines and has been implicated in spatial learning and hippocampal plasticity. Genetic variation in rs12807809 in the NRGN gene has recently been confirmed to be associated with schizophrenia in a meta-analysis of genome-wide association studies: the T-allele was found to be genome-wide significantly associated with schizophrenia. Cognitive tests and personality questionnaires were administered in a large sample of healthy subjects. Brain activation was measured with functional magnetic resonance imaging (fMRI) during an episodic memory encoding and retrieval task in a subsample. All subjects were genotyped for NRGN rs12807809. There was no effect of genotype on personality or cognitive measures in the large sample. Homozygote carriers of the T-allele showed better performance in the retrieval task during fMRI. After controlling for memory performance, differential brain activation was evident in the anterior cingulate cortex for the encoding and posterior cingulate regions during retrieval. We could demonstrate that rs12807809 of NRGN is associated with differential neural functioning in the anterior and posterior cingulate. These areas are involved in episodic memory processes and have been implicated in the pathophysiology of schizophrenia in structural and functional imaging as well as postmortem studies.

Proteomics analysis of the expression of neurogranin in murine neuroblastoma (Neuro-2a) cells reveals its involvement for cell differentiation.

Neurogranin (Ng) is a neural-specific, calmodulin (CaM)-binding protein that is phosphorylated by protein kinase C (PKC). Although its biochemical property has been well characterized, the physiological function of Ng needs to be elucidated. In the present study, we performed proteomics analysis of the induced compositional changes due to the expression of Ng in murine neuroblastoma (Neuro-2a) cells using isotope coded affinity tags (ICAT) combined with 2-dimensional liquid chromatography/tandem mass spectrometry (2D-LC/MS/MS). We found that 40% of identified proteins were down-regulated and most of these proteins are microtubule components and associated proteins that mediated neurite outgrowth. Western blot experiments confirmed the expression of alpha-tubulin and microtubule- associated protein 1B (MAP 1B) was dramatically reduced in Neuro-2a-Ng cells compared to control. Cell morphology of Neuro-2a-Ng showed far less neurites than the control. Serum deprivation induced the extension of only one or two long neurites per cell in Neuro-2a-Ng, contrasting to the extension of multiple neurites per control cell. Ng may be linked to neurite formation by affecting expression of several microtubule related proteins. Furthermore, the PKC activator (PMA) induced an enhanced ERK1/2 activity in the cells that expressed Ng. The mutation of Ng at S36A caused sustained increase of ERK1/2 activity, whereas the ERK1/2 activity in mutation at I33Q showed no difference compared to wild type Ng, suggesting the phosphorylation of Ng but not the CaM /Ng interaction plays an important role in ERK activation. Ng may be involved in neuronal growth and differentiation via PKC and ERK1/2 signaling pathways.

Characterization of transcriptional regulation of neurogranin by nitric oxide and the role of neurogranin in SNP-induced cell death: implication of neurogranin in an increased neuronal susceptibility to oxidative stress.

Neurogranin (Ng), a calmodulin (CaM)-binding protein kinase C (PKC) substrate, regulates the availability of Ca(2+)/CaM complex and modulates the homeostasis of intracellular calcium in neurons. Previous work showed Ng oxidation by NO donor induces increase in [Ca(2+)](i). The current study demonstrated that the gene transcription of Ng could be up-regulated by various nitric oxide (NO) donors via a NO-soluble guanylyl cyclase (sGC)-mediated pathway. Furthermore, ectopic expression of neuronal nitric oxide synthase (nNOS) in human embryonic kidney 293 cells (HEK 293) exhibited a nNOS-concentration-dependent biphasic regulatory effect on Ng gene transcription. One of the NO donors, sodium nitroprusside (SNP), however, induced cell death of neuroblastoma Neuro-2a cells. The potency of SNP-induced cell death was shown to be higher in Neuro-2a cells expressing recombinant Ng, as compared with Neuro-2a control cells without Ng expression in cell viability and apoptosis assays. Single-cell fluorescence imaging and site-directed mutagenesis studies suggest that Ng promotes SNP-induced cell death through an amplification of calcium-mediated signaling, which requires the interaction between CaM and IQ motif of Ng. Increased neuronal susceptibility rendered by Ng in response to pathophysiological NO production is suggested to be involved in the selective vulnerability of neurons to oxidative insults in the CNS.

Disruption of acute opioid dependence by antisense oligodeoxynucleotides targeting neurogranin.

Neurogranin has been suggested to serve as a common regulator synchronizing the activities of PKC and CaMKII in acute opioid tolerance. To examine if a similar mechanism exists in acute opioid dependence, we directly targeted neurogranin using antisense oligodeoxynucleotides. Male ICR mice were pretreated with neurogranin antisense or mismatch oligodeoxynucleotides (2 microg/day, i.c.v.) for 3 consecutive days. On Day 4, morphine (100 mg/kg, s.c.) was used to induce dependence, as revealed by naloxone-precipitated withdrawal in saline or mismatch-pretreated mice. Antisense-pretreated mice showed decreased neurogranin expression, lack of morphine-induced phosphorylation of neurogranin and activation of CaMKII and CREB, and absence of naloxone-induced withdrawal jumping. Taken together, these data suggest that neurogranin plays an essential role in acute opioid dependence, possibly by affecting the CaMKII and CREB signaling pathway.

Neurogranin binds to phosphatidic acid and associates to cellular membranes.

Neurogranin (Ng) is a 78-amino-acid-long protein concentrated at dendritic spines of forebrain neurons that is involved in synaptic plasticity through the regulation of CaM (calmodulin)-mediated signalling. Ng features a central IQ motif that mediates binding to CaM and is phosphorylated by PKC (protein kinase C). We have analysed the subcellular distribution of Ng and found that it associates to cellular membranes in rat brain. In vitro binding assays revealed that Ng selectively binds to PA (phosphatidic acid) and that this interaction is prevented by CaM and PKC phosphorylation. Using the peptide Ng-(29-47) and a mutant with an internal deletion (Ng-IQless), we have shown that Ng binding to PA and to cellular membranes is mediated by its IQ motif. Ng expressed in NIH-3T3 cells accumulates at peripheral regions of the plasma membrane and localizes at intracellular vesicles that can be clearly visualized following saponin permeabilization. This distribution was affected by PLD (phospholipase D) and PIP5K (phosphatidylinositol 4-phosphate 5-kinase) overexpression. Based on these results, we propose that Ng binding to PA may be involved in Ng accumulation at dendritic spines and that Ng could modulate PA signalling in the postsynaptic environment.

Synaptic plasticity and phosphorylation.

A number of neuronal functions, including synaptic plasticity, depend on proper regulation of synaptic proteins, many of which can be rapidly regulated by phosphorylation. Neuronal activity controls the function of these synaptic proteins by exquisitely regulating the balance of various protein kinase and protein phosphatase activity. Recent understanding of synaptic plasticity mechanisms underscores important roles that these synaptic phosphoproteins play in regulating both pre- and post-synaptic functions. This review will focus on key postsynaptic phosphoproteins that have been implicated to play a role in synaptic plasticity.

Role of the neurogranin concentrated in spines in the induction of long-term potentiation.

Synaptic plasticity in CA1 hippocampal neurons depends on Ca2+ elevation and the resulting activation of calmodulin-dependent enzymes. Induction of long-term depression (LTD) depends on calcineurin, whereas long-term potentiation (LTP) depends on Ca2+/calmodulin-dependent protein kinase II (CaMKII). The concentration of calmodulin in neurons is considerably less than the total concentration of the apocalmodulin-binding proteins neurogranin and GAP-43, resulting in a low level of free calmodulin in the resting state. Neurogranin is highly concentrated in dendritic spines. To elucidate the role of neurogranin in synaptic plasticity, we constructed a computational model with emphasis on the interaction of calmodulin with neurogranin, calcineurin, and CaMKII. The model shows how the Ca2+ transients that occur during LTD or LTP induction affect calmodulin and how the resulting activation of calcineurin and CaMKII affects AMPA receptor-mediated transmission. In the model, knockout of neurogranin strongly diminishes the LTP induced by a single 100 Hz, 1 s tetanus and slightly enhances LTD, in accord with experimental data. Our simulations show that exchange of calmodulin between a spine and its parent dendrite is limited. Therefore, inducing LTP with a short tetanus requires calmodulin stored in spines in the form of rapidly dissociating calmodulin-neurogranin complexes.

Attenuation of opioid tolerance by antisense oligodeoxynucleotides targeting neurogranin.

Neurogranin is capable of regulating protein kinase C and Ca2+/calmodulin-dependent protein kinase II. In this study, we examined the role of neurogranin in opioid tolerance. Increased phosphorylation of neurogranin was found in opioid tolerance. Opioid tolerance was absent in morphine (100 mg/kg)-treated mice that were also pretreated with neurogranin antisense oligodeoxynucleotides (2 microg/day, i.c.v. for 3 days). The behavioral effect correlated with the decreased expression of neurogranin. These data suggest that neurogranin may be critical in the development of opioid tolerance.