Phosphorylation of Ca2+/calmodulin-dependent protein kinase type ii and the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (ampa) receptor in response to a threonine-devoid diet

doi:10.1016/j.neuroscience.2004.03.066

Neuroscience

Volume 126, Issue 4, 2004, Pages 1053-1062

https://doi.org/10.1016/j.neuroscience.2004.03.066 Get rights and content

Abstract

The anterior piriform cortex (APC) functions as a chemosensor for indispensable amino acid deficiency and responds to this deficiency with increased activity, as indicated by observations including averaged evoked-potentials and c-fos expression in the APC. Little is known of the intracellular signaling mechanisms that mediate this deficiency-related increase in neuronal excitability, but previous studies have shown effects on intracellular Ca²⁺ in deficient APC slices in vitro. In the present study we hypothesized that indispensable amino acid deficiency increases intraneuronal Ca²⁺, resulting in autophosphorylation of calcium/calmodulin-dependent protein kinase type II (CaMKII) in vivo. Results demonstrated that phosphorylation levels of CaMKII (pCaMKII) in APC neurons increased at 20 and 40 min after a single meal of threonine-devoid diet. Phosphorylation of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit (GluR1) at the serine 831 (S831) site was modestly increased in the APC in response to a threonine-devoid meal. The GluR1 subunit also showed increased phosphorylation at the 845 (S845) site, suggesting additional signaling mechanisms. Although phosphorylation of CaMKII was sustained, phosphorylation of the GluR1 subunit returned to control levels by 40 min. These effects of amino acid deficiency did not occur throughout the brain as neither CaMKII nor GluR1 showed increased phosphorylation in the neocortex.

These findings support the notion that calcium and glutamate signaling in the APC, but not throughout the brain, are triggered during early responses to amino acid deficiency. They also suggest that longer-term changes in APC neurons in response to such a deficiency may be mediated at least in part by CaMKII.

Section snippets

Experimental diets

Diets were made of with purified l-amino acids (Ajinomoto Inc., Teaneck, NJ, USA) as the sole source of amino acids, and the diets have previously been described in detail (Koehnle et al., 2003). The basal diet contained 12.4% total amino acids with 4.8% (wt:wt) of IAAs, which provided 75% of IAA requirements for maximum growth except for threonine, which was included at 50% of its requirement. The basal threonine-devoid (BTD) diet was analogous to the basal diet except that it completely

Phosphorylated CaMKII

The α subunit of CaMKII (αCaMKII) showed increased phosphorylation (at threonine 286) in the APC, APC_lot, and APC_vr in response to a BTD diet at 20 min, which was sustained over a 40-min period. Fig. 1A, B shows results for αCaMKII and p-αCaMKII in the APC after a BTD or basal meal. The same amount of tissue protein was added to each gel well and each treatment was replicated within a gel, as described above. Visual inspection of the Western blot bands demonstrates an increased amount of

Discussion

In the present study rats were conditioned for 7 days on basal diet, then after an overnight fast they were fed either a basal or BTD meal. Previous studies have shown that the APC serves as a chemosensory area for IAA deficiency (Beverly et al., 1990, Blevins et al., 2000, Gietzen et al., 1986, Gietzen, 1993, Russell et al., 2003) and that the APC responds to this deficiency with increased excitability, as shown by an increased averaged-evoked potential (Hasan et al., 1998), increased c-fos

Acknowledgements

This research was supported by grants from the National Institutes of Health (NS53749, NS33347, and NS43210), and the US Department of Agriculture (NR1CRGP 2000-01049).

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Low protein amounts are used in ketogenic diets (KDs), where an essential (indispensable) amino acid (IAA) can become limiting. Because the chemically sensitive, seizurogenic, anterior piriform cortex (APC) is excited by IAA limitation, an imbalanced KD could exacerbate seizure activity.
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Adult rats showed increased susceptibility to seizures in both chemical (58%) and electroshock (doubled) testing after 7 d on a ThrDev diet compared with CD (each trial, P ≤ 0.05). Seizure-prone Mongolian gerbils had fewer seizures after receiving a KD, but exacerbated seizures (68%) after 1 meal of KD minus Thr (KD–T compared with CD, P < 0.05). In kindled rats fed KD–T, both counts (19%) and severities (77%) of seizures were significantly elevated (KD–T compared with CD, P < 0.05).
Gene transcript changes were consistent with enhanced seizure susceptibility (7–21 net-fold increases, P = 0.045–0.001) and glutamate release into the APC was increased acutely (4-fold at 20 min, 2.6-fold at 60 min, P < 0.05) after 7 d on a ThrDev diet.
Seizure severity in rats and gerbils was reduced after KDs and exacerbated by ThrDev, both in KD- and CD-fed animals, consistent with the mechanistic studies. We suggest that a complete protein profile in KDs may improve IAA balance in the APC, thereby lowering the risk of seizures.
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Moreover, experiments on cultured hippocampal neurons showed that the significant increase in spine and protrusion density caused by naloxone treatment is blocked in cells transfected with dominant-negative mutant calcium/calmodulin-dependent protein kinase II (CaMKII) plasmids (48). In the cortex, the activation of CaMKII as well as other kinases has been shown to be involved in GluA1-S845 phosphorylation upregulation mechanisms (49,50). Thus, CaMKII may play a role in the mechanisms behind the increased GluA1 membrane expression by naltrexone.
The opioid antagonists naloxone/naltrexone are involved in improving learning and memory, but their cellular and molecular mechanisms remain unknown. We investigated the effect of naloxone/naltrexone on hippocampal α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) trafficking, a molecular substrate of learning and memory, as a probable mechanism for the antagonists activity.
To measure naloxone/naltrexone-regulated AMPAR trafficking, pHluorin-GluA1 imaging and biochemical analyses were performed on primary hippocampal neurons. To establish the in vivo role of GluA1-Serine 845 (S845) phosphorylation on the behavioral effect induced by inhibition of the endogenous μ-opioid receptor (MOR) by naltrexone, MOR knockout, and GluA1-S845A mutant (in which Ser⁸⁴⁵ was mutated to Ala) mice were tested in a water maze after chronic naltrexone administration. Behavioral responses and GluA1 levels in the hippocampal postsynaptic density in wild-type and GluA1-S845A mutant mice were compared using western blot analysis.
In vitro prolonged naloxone/naltrexone exposure significantly increased synaptic and extrasynaptic GluA1 membrane expression as well as GluA1-S845 phosphorylation. In the MOR knockout and GluA1-S845A mutant mice, naltrexone did not improve learning, which suggests that naltrexone acts via inhibition of endogenous MOR action and alteration of GluA1 phosphorylation. Naltrexone-treated wild-type mice had significantly increased phosphorylated GluA1-S845 and GluA1 levels in their hippocampal postsynaptic density on the third day of acquisition, which is the time when naltrexone significantly improved learning.
The beneficial effect of naltrexone on spatial learning and memory under normal conditions appears to be the result of increasing GluA1-S845 phosphorylation-dependent AMPAR trafficking. These results can be further explored in a mouse model of memory loss.
Ca<sup>2+</sup>/calmodulin-dependent protein kinase II inhibitors disrupt AKAP79-dependent PKC signaling to GluA1 AMPA receptors
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AKAP79/150 thus not only restricts PKC sensitivity to some PKC-selective reagents but as illustrated here also endows PKC with pharmacological sensitivity to classical CaMKII inhibitors that do not normally target PKC. Enhancement of GluA1 phosphorylation at Ser-831 is linked with LTP and is also a downstream consequence of other manipulations that alter synaptic function (56–72). KN-62 or KN-93 sensitivity and/or CaMKII autophosphorylation at Thr-286 has been used as central criteria for CaMKII involvement in these phenomena (57, 61, 62, 68, 70–72).
GluA1 (formerly GluR1) AMPA receptor subunit phosphorylation at Ser-831 is an early biochemical marker for long-term potentiation and learning. This site is a substrate for Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) and protein kinase C (PKC). By directing PKC to GluA1, A-kinase anchoring protein 79 (AKAP79) facilitates Ser-831 phosphorylation and makes PKC a more potent regulator of GluA1 than CaMKII. PKC and CaM bind to residues 31–52 of AKAP79 in a competitive manner. Here, we demonstrate that common CaMKII inhibitors alter PKC and CaM interactions with AKAP79(31–52). Most notably, the classical CaMKII inhibitors KN-93 and KN-62 potently enhanced the association of CaM to AKAP79(31–52) in the absence (apoCaM) but not the presence of Ca²⁺. In contrast, apoCaM association to AKAP79(31–52) was unaffected by the control compound KN-92 or a mechanistically distinct CaMKII inhibitor (CaMKIINtide). In vitro studies demonstrated that KN-62 and KN-93, but not the other compounds, led to apoCaM-dependent displacement of PKC from AKAP79(31–52). In the absence of CaMKII activation, complementary cellular studies revealed that KN-62 and KN-93, but not KN-92 or CaMKIINtide, inhibited PKC-mediated phosphorylation of GluA1 in hippocampal neurons as well as AKAP79-dependent PKC-mediated augmentation of recombinant GluA1 currents. Buffering cellular CaM attenuated the ability of KN-62 and KN-93 to inhibit AKAP79-anchored PKC regulation of GluA1. Therefore, by favoring apoCaM binding to AKAP79, KN-62 and KN-93 derail the ability of AKAP79 to efficiently recruit PKC for regulation of GluA1. Thus, AKAP79 endows PKC with a pharmacological profile that overlaps with CaMKII.
The sensing of essential amino acid deficiency in the anterior piriform cortex, that requires the uncharged tRNA/GCN2 pathway, is sensitive to wortmannin but not rapamycin
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Animals detect and reject their first essential/indispensable amino acid (IAA) deficient meal within 20 min; this IAA sensing requires an intact anterior piriform cortex (APC). In the biochemical responses to IAA deficiency in the APC we have shown that: uncharged tRNA is the primary sensor; IAA transport is increased; and signaling, including the extracellular-regulated kinase (ERK1/2), is activated. The mammalian target of rapamycin (mTOR) is a potential AA sensor and is regulated by AA transport. Previously, the inhibitors, rapamycin for mTOR, wortmannin for phosphoinositide 3 kinase (PI3K) and PD98059 for ERK, each blocked the upregulation of the System A transporter in IAA depleted APC neurons. Here we injected these same inhibitors into the APC and measured intake of an IAA deficient diet. Rapamycin had no effect on the rejection of the IAA deficient diet, but wortmannin increased ERK activation and intake of the deficient diet before 40 min and PD98059 acted after 40 min to increase the second meal. While the specific wortmannin target involved in blocking the behavioral response remains unclear, we conclude that mTOR is dispensable for sensing IAA deficiency in the APC, and that ERK is associated with the secondary learned responses to IAA deficient diets.
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Indispensable amino acids are neither synthesized nor stored in animals and are rapidly depleted when not provided by the diet. To maintain homeostasis, organisms must sense deficiency of an indispensable amino acid and implement a repletion strategy. In rats and birds, the anterior piriform cortex houses the detector, but its mechanism has evaded description for >50 years. Recently, rapid detection of amino acid depletion was shown behaviorally when naïve animals, pre-fed a low nitrogen diet, terminated their first deficient meal within 20 min. The general amino acid control system of yeast, which is activated by amino acid deprivation via deacylated tRNA, was found to be active in rodent brain, showing conservation of amino acid sensory mechanisms across eukaryotic species.
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2004, Brain Research
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The glutamate receptors within the APC have been linked to involvement in the central control of essential AA feeding model [17]. Recent studies indicate that the alpha subunit of the GluR1 glutamate receptor is upregulated by 20 min in the APC [41]. As noted earlier, the glutamatergic pyramidal cells are the primary cells in the APC [20], and are clearly activated before 30 min, as shown by c-Jun in the nuclei [19].
Animals decrease intake of an indispensable amino acid (AA)-deficient or devoid diet, due in part to decreased dietary limiting AA (DLAA) concentrations within the anterior piriform cortex (APC), and to a recognition process that occurs as early as 20 min following exposure to AA deficiencies. Glutamate levels within the APC change in response to AA deficiencies. The APC projects to the lateral hypothalamus (LH), where glutamate acts to stimulate food intake. We hypothesize that the APC, through glutamatergic projections to the LH, inhibits the LH, which signals to reject the AA-deficient or devoid diet, and trigger aversions to the AA-deficient or devoid diet via an ascending pathway to the APC. We examined the effects of (1) bilateral APC and LH blockade of glutamate's NMDA receptors with the antagonist, D-AP5, (2) APC blockade of AMPA receptors with the antagonist, NBQX, to block glutamate transmission from the APC, and (3) direct injection of the agonist, NMDA, into the LH on intake of the AA-deficient, devoid, or corrected diet. Administration of D-AP5 into the APC increased intake of AA-deficient diet by 6 h, but D-AP5 in the LH decreased AA-devoid diet preferentially over AA corrected intake sooner. NBQX in the APC increased AA-deficient diet intake, also at 6 h. NMDA injection into the LH-stimulated intake of the AA corrected diet by 3 h, but did not affect AA-devoid diet intake. Thus, the glutamate receptors in the APC and LH are involved in the feeding responses to AA-deficient diet, albeit with regional differences. We suggest that glutamate mediates the anorectic responses to AA-deficient diets through recognition of AA-devoid diet with the glutamatergic output cells of the APC sending glutamate-based signals for changes in food intake within the LH and through learned avoidance of AA-deficient diet within the APC, as indicated through the more immediate and prolonged periods of activation within the LH and APC, respectively.

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Phosphorylation of Ca2+/calmodulin-dependent protein kinase type ii and the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (ampa) receptor in response to a threonine-devoid diet

Abstract

Section snippets

Experimental diets

Phosphorylated CaMKII

Discussion

Acknowledgements

J Biol Chem

Brain Res

J Biol Chem

Biophys J

J Nutr

J Nutr

J Nutr

Neuron

J Neurosci Methods

J Nutr

Physiol Behav

J Biol Chem

J Nutr

Mol Brain Res

Neuron

Mol Brain Res

Physiol Behav

The initial recognition phase. Brain Res

Control of GluR1 AMPA receptor function by cAMP-dependent protein kinase

J Neurosci

Integrins regulate DLG/FAS2 via a CaM kinase II-dependent pathway to mediate synapse elaboration and stabilization during postembryonic development

Development

Effect of dietary limiting amino acid in prepyriform cortex on food intake

Am J Physiol

CaMKII-dependent phosphorylation of NR2A and NR2B is decreased in animals characterized by hippocampal damage and impaired LTP

Eur J Neurosci

Regulation of AMPA receptors by phosphorylation

Neurochem Res

Is persistent activity of calcium/calmodulin-dependent kinase required for the maintenance of LTP?

J Neurophysiol

Phosphorylation of Ca²⁺/calmodulin-dependent protein kinase type ii and the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (ampa) receptor in response to a threonine-devoid diet