Deletion of CaMKIIα disrupts glucose metabolism, glutamate uptake, and synaptic energetics in the cerebral cortex

Ca2+/calmodulin‐dependent protein kinase II alpha (CaMKIIα) is a key regulator of neuronal signaling and synaptic plasticity. Synaptic activity and neurotransmitter homeostasis are closely coupled to the energy metabolism of both neurons and astrocytes. However, whether CaMKIIα function is implicated in brain energy and neurotransmitter metabolism remains unclear. Here, we explored the metabolic consequences of CaMKIIα deletion in the cerebral cortex using a genetic CaMKIIα knockout (KO) mouse. Energy and neurotransmitter metabolism was functionally investigated in acutely isolated cerebral cortical slices using stable 13C isotope tracing, whereas the metabolic function of synaptosomes was assessed by the rates of glycolytic activity and mitochondrial respiration. The oxidative metabolism of [U‐13C]glucose was extensively reduced in cerebral cortical slices of the CaMKIIα KO mice. In contrast, metabolism of [1,2‐13C]acetate, primarily reflecting astrocyte metabolism, was unaffected. Cellular uptake, and subsequent metabolism, of [U‐13C]glutamate was decreased in cerebral cortical slices of CaMKIIα KO mice, whereas uptake and metabolism of [U‐13C]GABA were unaffected, suggesting selective metabolic impairments of the excitatory system. Synaptic metabolic function was maintained during resting conditions in isolated synaptosomes from CaMKIIα KO mice, but both the glycolytic and mitochondrial capacities became insufficient when the synaptosomes were metabolically challenged. Collectively, this study shows that global deletion of CaMKIIα significantly impairs cellular energy and neurotransmitter metabolism, particularly of neurons, suggesting a metabolic role of CaMKIIα signaling in the brain.


| INTRODUC TI ON
The brain is a highly energy-consuming organ.Most of the energy spent by the brain is used to maintain neuronal signaling and the cerebral metabolic rate is closely correlated to synaptic activity (Sibson et al., 1998;Yu et al., 2018).The brain primarily relies on glucose oxidation for energy production; however, several other substrates, including neurotransmitters, are also used as cerebral fuels (Dienel, 2019;Mergenthaler et al., 2013).Excitatory glutamatergic neurons are particularly energy demanding, but other brain cells, such as astrocytes and inhibitory neurons, also require a steady provision of energy substrates (Yu et al., 2018).Astrocytes are the primary homeostatic glial cell of the brain and function in close metabolic collaboration with both excitatory and inhibitory neurons (Andersen et al., 2023;Bonvento & Bolaños, 2021).A large fraction of synaptic glutamate and γ-aminobutyric acid (GABA), being the primary excitatory and inhibitory neurotransmitters, respectively, are taken up by astrocytes (Zhou & Danbolt, 2013).In the astrocytes, both glutamate and GABA are metabolized (Andersen et al., 2020;McKenna, 2012), which support glutamine synthesis.
Glutamine is subsequently transferred from astrocytes to neurons for the replenishment of the glutamate and GABA pools (Andersen & Schousboe, 2022;Peng et al., 1993).The exchange of neuronal glutamate/GABA and astrocytic glutamine is collectively termed the glutamate/GABA-glutamine cycle and is central for sustaining neurotransmission (Bak et al., 2006;Tani et al., 2014).This neurotransmitter recycling is further closely linked to the cellular energy metabolism of both neurons and astrocytes (Andersen, Markussen, et al., 2021;Schousboe et al., 2013).
The ability to regulate synapse strength is the molecular basis for learning and memory.In this regard, the synaptic enzyme Ca 2+ / calmodulin-dependent protein kinase II alpha (CaMKIIα) is a key regulator of neuronal signaling (Coultrap & Bayer, 2012;Yasuda et al., 2022).CaMKIIα is highly abundant in the brain and is particularly enriched in the forebrain, including the cerebral cortex and hippocampus (Erondu & Kennedy, 1985).At the subcellular level, CaMKIIα is located in neurons and is recruited to the post-synaptic densities of excitatory synapses upon high-frequency stimulation (Chapman et al., 2022).Here, CaMKIIα phosphorylates multiple synaptic proteins, including α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartate (NMDA)-type glutamate receptors, hereby modulating long-term potentiation (LTP) and synaptic plasticity (Coultrap & Bayer, 2012).However, at lower signaling intensities, CaMKIIα is also involved in the regulation of GABAergic signaling at inhibitory synapses, for instance by modulating GABA A and GABA B receptor activity (Guetg et al., 2010;Marsden et al., 2010).Several mutations of CaMKIIα have been shown to cause severe encephalopathies (Chia et al., 2018;Stephenson et al., 2017), and CaMKIIα dysregulation has been associated with neurodegenerative diseases (Ghosh & Giese, 2015).Given that CaMKIIα is critical for synaptic function, which is closely connected to energy metabolism, we asked how CaMKIIα function may be related to the cellular energetics of the brain.
To investigate the cerebral metabolic role of CaMKIIα, we applied a genetically engineered CaMKIIα knockout (KO) mouse.These mice display no gross anatomical defects of the central nervous system, but have impaired long-term potentiation and weakened spatial learning (Elgersma et al., 2004;Silva, Paylor, et al., 1992;Silva, Stevens, et al., 1992).Additionally, CaMKIIα KO mice display decreased sleep duration (Tatsuki et al., 2016), which could be affecting metabolic processes (Ode & Ueda, 2020).However, how the synaptic deficits may translate into, or potentially be causal in relation to, metabolic changes are unknown.Here, we assessed the metabolic function of CaMKIIα mice in acutely isolated brain slices by isotope tracing of 13 C enriched substrates and investigated synaptic energetics in isolated synaptosomes by measurements of glycolytic capacity and mitochondrial respiration.Our results reveal that mice deficient of CaMKIIα display impaired cerebral glucose metabolism and glutamate uptake, whereas synaptic metabolism became insufficient selectively during stimulation.The study highlights a critical role for CaMKIIα in relation to cerebral energetics and glutamate homeostasis, which may be important in several pathological conditions, including stroke and neurodegenerative disorders.

| Animals
Heterozygous CaMKIIα knockout (KO) mice (Camk2a −/+ ) were obtained from a founder colony at the Department of Neuroscience, Erasmus University Medical Center, Rotterdam, the Netherlands (a kind gift from Y. Elgersma).The mice were backcrossed onto a C57Bl6/J background, and a colony was maintained at the Department of Drug Design and Pharmacology, University of Copenhagen, Denmark.Homozygous CaMKIIα KO mice (Camk2a −/− , Camk2a tm3Sva , MGI: 2389262) (Elgersma et al., 2002) and corresponding wild-type littermates (Camk2a +/+ ) were generated by mating of heterozygotes.Mice were housed together in individually ventilated cages in a specific pathogen-free, humidity, and temperature-controlled facility with 12-h light/dark cycle and free access to water and chow.Experiments were performed throughout the 12-h light cycle of the animals.Camk2a −/− mice, hereafter referred to as CaMKIIα KO, were used at 12-14 weeks of age and corresponding wild-type littermates (Camk2a +/+ ) were used as controls (referred to as controls).In total, 41 mice were used for this study (22 controls and 19 KOs as indicated below).
Since sex hormones can affect brain mitochondrial function (Torrens-Mas et al., 2020), only male mice were used in this study and any potential sexspecific aspects of the work could therefore not be evaluated.

| Brain slice incubations
Incubation of acutely isolated brain slices was performed as reported previously (McNair et al., 2017).Mice ( 6 Lactate released from brain slices to the medium during incubation was determined by an L-lactic acid kit (Boehringer Mannheim/R-Biopharm/ Roche) according to the manufacturer's instructions.

| Isotope tracing using gas chromatographymass spectrometry (GC-MS) analysis
To determine the 13 C enrichment of TCA cycle intermediates and connected amino acids, brain slice extracts were analyzed by GC-MS as described previously (Walls et al., 2014).Briefly, extracts were reconstituted in water, acidified, and extracted twice with ethanol and metabolites derivatized using N-tert-butyldimethylsilyl-Nmethyltrifluoroacetamide.Samples were analyzed by GC (Agilent Technologies, 7820A, J&W GC column HP-5 MS) coupled to MS (Agilent Technologies, 5977E).The isotopic enrichment was corrected for the natural abundance of 13 C by analyzing standards containing the unlabeled metabolites of interest.Data from [U-13 C]glucose metabolism describing glycolytic activity (Figure 1) is presented as M + X, where M is the molecular ion and X is the number of 13 C atoms in the molecule.
F I G U R E 1 Glycolytic activity is unaffected by CaMKIIα deletion.Intracellular 13 C enrichments of alanine and lactate and total amounts of released lactate from cerebral cortical slices of wild-type control (black bars) and CaMKIIα knockout (KO) mice (blue bars) following metabolism of [U-13 C]glucose.[U-13 C]Glucose was provided at a concentration of 5 mM.Cellular metabolism of [U-13 C]glucose via glycolysis will lead to M + 3 labeling alanine and lactate, through the enzymes alanine aminotransferase (ALAT) and lactate dehydrogenase (LDH).The 13 C enrichment in alanine and lactate hereby reflects the glycolytic activity.Full statistical report can be found in Supplementary File 2.
Mean ± SEM, n = 6 from individual animals, Welch's two-tailed t-test or Mann-Whitney test, both with Benjamini-Hochberg correction.The remaining data from [U-13 C]glucose metabolism (Figure 2) and [1,2-13 C]acetate metabolism (Figure 3) is presented as the molecular carbon labeling (MCL), which is a weighted average of 13 C accumulation of a given metabolite (Andersen, Christensen, et al., 2021).Data from the [U-13 C]glutamate metabolism (Figure 4) and [U-13 C]GABA metabolism glutamate and [U-13 C]GABA metabolism can be found in (Andersen et al., 2017) and in Supplementary File 1, respectively.

| Determination of amino acid amounts by high-performance liquid chromatography (HPLC) analysis
To obtain fresh cerebral cortical tissue, mice (10 controls & 7 KOs) were killed, one at a time, by cervical dislocation, decapitated, and the brain excised.The cerebral cortex was dissected on ice and the tissue quickly frozen at −80°C.The tissue was subsequently thawed in ice-cold 70% ethanol, sonicated, and centrifuged (4000 g × 20 min), and the supernatant was lyophilized before further analysis.Pellets were saved for protein determination by

| Isolation of synaptosomes and determination of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)
Isolation and analysis of cerebral cortical synaptosomes were performed as described previously (Andersen, Skotte, et al., 2021).One control and one CaMKIIα KO mouse (in total 6 controls & 6 KOs) were killed in tandem by cervical dislocation and decapitated, and the brains were dissected on ice.All procedures were performed on ice or at 4°C.Synaptosomes were isolated using a Percoll gradient, and protein amounts were determined by the Bradford method.The oxygen rate (OCR, pmol O 2 /min) and extracellular acidification rate (ECAR, mpH/min) of the isolated regional synaptosomes were assessed at 37°C using a Seahorse XFe96 analyzer (Seahorse Biosciences) with a protocol adapted from (Hohnholt et al., 2017).

| Unchanged neurochemical profile
Given the widespread expression of CaMKIIα in the brain, we first asked whether deletion of this abundant enzyme would lead to alterations of the general neurochemical profile of the cerebral cortex.Accordingly, we quantified a panel of central amino acids in freshly isolated cerebral cortical tissue of CaMKIIα KO and control mice (Table 1).The intracellular levels of all investigated amino acids were unchanged in the brain tissue, demonstrating that CaMKIIα deletion does not disrupt the homeostasis of these cerebral amino acids.

| Cerebral glycolytic activity is maintained
Next, we sought to functionally assess the potential effects of CaMKIIα deletion on brain energy metabolism.To this end, we performed stable slices (p = 0.47) (Figure 1), suggesting that the glycolytic activity is maintained in the cerebral cortex of CaMKIIα KO mice.

| Reduced oxidative metabolism of glucose
In

| Astrocyte acetate metabolism is sustained
Given the extensive neurotransmitter recycling between neurons and astrocytes, the energy metabolism of astrocytes is closely connected to neuronal function.To functionally probe astrocytes energetics, cerebral cortical slices of CaMKIIα KO and control mice were incubated in the presence of [1,2-13 C]acetate.Acetate is primarily metabolized in astrocytes and enters the TCA cycle as acetyl CoA M + 2 units (Figure 3).No significant differences were observed in the TCA cycle intermediates citrate (p = 0.27), α-ketoglutarate (p = 0.10), fumarate (p = 0.73), and malate (p = 0.07) from metabolism of [1,2-13 C]acetate in the slices of CaMKIIα KO mice (Figure 3).This was also the case of the connected amino acids aspartate (p = 0.31), glutamate (p = 0.24), glutamine (p = 0.10), and GABA (p = 0.07).These results suggest that deletion of CaMKIIα does not affect the metabolic capacity or synthesis of amino acids generated from acetate metabolism in astrocytes.

| Impaired uptake and metabolism of glutamate
The primary excitatory neurotransmitter, glutamate, is closely linked to cellular energy metabolism of the brain.To investigate functional glutamate uptake and metabolism, we next incubated cerebral cortical slices of CaMKIIα KO mice in the presence of [U-13 C]glutamate (Figure 4).Quantification of the total amounts of intracellular glutamate in the slices after incubation showed no differences in glutamate levels in the CaMKIIα KO slices (controls: 541.7 ± 91.5 nmol/mg protein, n = 6 vs. KO: 429.6 ± 47.9 nmol/mg protein, n = 6, p = 0.31).Uptake of [U-13 C]glutamate into the slices will lead to enrichment of glutamate M + 5, which was decreased in the CaMKIIα KO slices (−17.5%,p = 0.042) (Figure 4 These results suggest a reduced glutamate uptake capacity in the CaMKIIα KO cerebral cortex, consistently reflected as a lower 13 C enrichment of the connected metabolites.

| Oxidative metabolism of GABA is unaffected
We recently showed that cerebral GABA metabolism is more active than previously assumed (Andersen et al., 2020).Since CaMKIIα also regulates the activity of inhibitory GABAergic synapses, brain GABA uptake and metabolism were investigated by incubating the cerebral cortical slices in the presence of [U-13 C] GABA (Figure 5).The total amounts of GABA after incubation were unchanged in the slices (controls: 109.9 ± 16.7 nmol/mg protein, n = 6 vs. KO: 98.0 ± 11.8 nmol/mg protein, n = 6, p = 0.57).In addition, the unaffected 13 C enrichment of GABA M + 4 (p = 0.68) suggests a maintained capacity of GABA uptake (Figure 5).

| Disrupted synaptic mitochondrial function and glycolytic capacity
Finally, given that CaMKIIα is a synaptic enzyme, we addressed how CaMKIIα deletion may affect synaptic energetics.To test this, we assessed the mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of isolated synaptosomes of the CaMKIIα KO cerebral cortex (Figure 6a).The synaptosomes were provided with three different substrate combinations (Figure 6a) and challenged by sequential addition of metabolic modulators allowing for the assessment of mitochondrial function (Figure 6b).
In the presence of glucose as the only respiratory substrate, synaptosomes of CaMKIIα KO mice displayed maintained OCRs in the basal state (p = 0.69) and upon sodium channel activation by veratridine (p = 0.051) (Figure 6c).However, a lower proton leak (−16.5%,p = 0.019) and uncoupled OCR (−26.3%, p = 0.005) were found in the CaMKIIα KO synaptosomes.To boost the mitochondrial respiration, pyruvate was added to glucose as respiratory substrate (Figure 6d).
In the presence of pyruvate, CaMKIIα KO synaptosomes again displayed a maintained basal OCR (p = 0.19).However, the OCR was reduced during sodium channel stimulation (−23.6%,p = 0.006), and a lower proton leak (−22.0%,p = 0.005) and uncoupled OCR (−27.4%, p = 0.004) were observed in CaMKIIα KO mice (Figure 6d).The synaptosomes were also provided with glutamate in addition to glucose (Figure 6e).As observed for the other substrate combinations, the basal OCR of the synaptosomes was unchanged in the presence of glutamate (p = 0.31).However, the OCR upon veratridine addition was reduced when glutamate was present (−20.5%,p = 0.014), whereas the proton leak (p = 0.18) and uncoupled OCR (p = 0.13) were unaffected in the CaMKIIα KO synaptosomes.
Alongside the OCR measurements, the release of lactate from the synaptosomes was assessed as the extracellular acidification rate (ECAR), which serves a measure of glycolytic activity (Figure 6a).
In line with the maintained glycolytic activity of the brain slices after [U-13 C]glucose metabolism (Figure 1), the synaptosomal ECAR was unchanged when provided with glucose as respiratory substrate, both in the basal state and during sodium channel activation (Figure 6f).A sustained basal ECAR of the synaptosomes was also observed when pyruvate (Figure 6g) and glutamate (Figure 6h) were added as substrates.However, the synaptosomal glycolytic capacity was reduced upon veratridine addition when pyruvate (Figure 6g)

| DISCUSS ION
In this study, we show that global deletion of the central synaptic enzyme CaMKIIα leads to widespread alterations of brain energy and neurotransmitter metabolism (summarized in Figure 7).Oxidative metabolism of glucose was extensively reduced in cerebral cortical brain slices of CaMKIIα KO mice, whereas astrocyte acetate and GABA metabolism were maintained.Impaired uptake, and subsequent metabolism, of glutamate was also observed in brain slices of the CaMKIIα KO mice.Finally, synaptic energetics were unaffected during resting conditions, but became insufficient during sodium channel stimulation and mitochondrial uncoupling.
Although CaMKIIα has been studied for decades (Bayer & Schulman, 2019), several aspects of its function, including its link to brain metabolism, remain to be uncovered.Deletion of CaMKIIα in mice impairs synaptic LTP and spatial learning, but does not affect gross body or brain physiology (Silva, Paylor, et al., 1992;Silva, Stevens, et al., 1992).Despite a maintained overall amino acid profile (Table 1) and glycolytic activity (Figure 1), oxidative glucose metabolism was greatly reduced in cerebral cortical slices of CaMKIIα KO mice (Figure 2).On the contrary, metabolism of the astrocyteselective substrate [1,2-13 C]acetate was maintained in the CaMKIIα KO brain (Figure 3).The unchanged astrocyte metabolism suggests that the large impairments of [U-13 C]glucose metabolism could be attributed to neuronal metabolism.This would correlate well with the predominant neuronal expression profile of CaMKIIα (Ochiishi et al., 1994).Furthermore, it is estimated that 80% of the brain's oxidative glucose metabolism is demanded by excitatory glutamatergic neurons (Yu et al., 2018).Additionally, the reduced 13 C enrichment from [U-13 C]glucose metabolism was least pronounced in citrate (−12.8%) and glutamine (−12.9%), which are markers of astrocyte metabolism in slices (McNair et al., 2017).Given that synaptic plasticity and memory formation are highly energy requiring processes (Karbowski, 2019;Plaçais et al., 2017), the observed reduced oxi- reported to regulate the activity of AMP-activated protein kinase (AMPK) (Raney & Turcotte, 2008), whereas CaMKII was suggested to regulate AMPK activity in the heart (Meng et al., 2022).In both instances, activation of the CaMKII-AMPK pathway was shown to regulate fatty acid metabolism (Meng et al., 2022;Raney & Turcotte, 2008).Indeed, AMPK serves as a master metabolic switch, which upon activation promotes catabolic pathways and ATP generation (Ronnett et al., 2009).AMPK is expressed in both neurons and astrocytes (Blázquez et al., 1999;Turnley et al., 1999), where it regulates glucose uptake, glycolytic activity, and TCA cycle capacity (Voss et al., 2020;Weisová et al., 2009).Diminished CaMKIIα signaling may therefore reduce AMPK activity and lead to lower glucose oxidation.Given the intimate relationship between brain energy metabolism and synaptic transmission, it is intriguing to speculate that AMPK may facilitate the increased metabolic demand of CaMKIIαmediated synaptic plasticity, but this has to be experimentally validated.Furthermore, CaMKIIα has also been reported to phosphorylate the transcription factor Forkhead box O3 (FOXO3) hereby increasing its transcriptional activity (Tao et al., 2013).FOXO3 is a potent regulator of several cellular processes including apoptosis, resistance to oxidative stress, and cellular metabolism (van der Horst & Burgering, 2007).Interestingly, FOXO3 activity is also regulated by AMPK activity (Greer et al., 2007), suggesting that deletion of CaMKIIα may cause a metabolic reprogramming via AMPK and FOXO3.In addition, it must be noted that four variants of CaMKII are expressed in the brain (α, β, δ, γ), with the α and β isoforms primarily located in neurons and the δ variant in astrocytes (Ochiishi et al., 1994;Sakagami et al., 2000;Takeuchi et al., 2000).Deletion of both the α and β isoforms of CaMKII in adult mice is lethal (Kool et al., 2019).This suggests that the CaMKII isoforms are able to com- Synapses utilize large amounts of energy, particularly during excitatory signaling.Here, we found that regardless of the provided substrate combinations, synaptosomal respiration, and glycolytic activity, were maintained during resting conditions in the CaMKIIα KO brain (Figure 6).However, when the synaptosomes were stimulated by sodium channel activation and mitochondrial uncoupling, to mimic the extensive energetic burden of synaptic signaling, the synaptic energetics of the CaMKIIα KO brains were not able to operate at the same levels as wild-type controls.A connection between mitochondrial function and CaMKII function is well established in the heart (Joiner et al., 2012;Luczak et al., 2020), but has been sparsely investigated in the brain.However, disruption of CaMKII activity was shown to perturb dynamin-related protein 1 (Drp1) function causing mitochondrial fragmentation in neurons of C. elegans (Jiang et al., 2015).This process is essential for neuronal plasticity, as dendritic mitochondria undergo F I G U R E 7 Summary of metabolic effects of CaMKIIα deletion.The metabolic consequences of CaMKIIα deletion were explored in the cerebral cortex of CaMKIIα knockout (KO) mice.Stable isotope tracing in brain slices of CaMKIIα KO mice revealed a maintained glycolytic activity, but a reduced capacity for oxidative glucose metabolism.Metabolism of acetate, which primarily reflects astrocyte metabolism, was, however, unaffected.Uptake and metabolism of the inhibitory neurotransmitter GABA were likewise maintained in the brain slices, whereas a significant reduction in uptake, and subsequent metabolism, of glutamate was found in the CaMKIIα KO mice.Isolated synaptosomes (nerve endings) of the CaMKIIα KO mice displayed intact basal glycolytic activity and mitochondrial respiration during resting conditions.However, when the synaptosomes were metabolically challenged by sodium channel activation and mitochondrial uncoupling, both the glycolytic and mitochondrial capacities became insufficient in the CaMKIIα KO synaptosomes.Collectively, the results of this study show that deletion of CaMKIIα impairs both energy and neurotransmitter metabolism, particularly of neurons, suggesting important metabolic roles of CaMKIIα signaling in the brain.
a rapid fission during LTP, which is directly regulated by CaMKII phosphorylation of Drp1 (Divakaruni et al., 2018).Furthermore, FOXO3 has also been found to induce mitochondrial biogenesis and respiration in an AMPK-dependent manner (Peserico et al., 2013) Neurotransmitter uptake is an essential process in regulating synaptic transmission, and transport systems of glutamate and GABA are expressed in both pre-synaptic neurons and astrocytes (Zhou & Danbolt, 2013).Here, we found that deletion of CaMKIIα selectively alters uptake and subsequent metabolism of glutamate (Figure 4), whereas GABA handling was unaffected (Figure 5).(Chawla et al., 2017), whereas pharmacological CaMKII inhibition disrupted surface expression of a splice variant of GLT-1 upon glutamate exposure in cultured astrocytes (Underhill et al., 2015).Although astrocytes take up the majority of synaptic glutamate (Danbolt, 2001), presynaptic neurons also express glutamate transporters mediating neuronal glutamate re-uptake (Rimmele & Rosenberg, 2016), which is important for energy metabolism and synaptic health (McNair et al., 2020;Rimmele et al., 2021).Given the neuronal expression profile of CaMKIIα and that CaMKII inhibition affects glutamate homeostasis in neurons (Ashpole et al., 2012), a neuronal contribution to the reduced glutamate uptake upon CaMKIIα deletion cannot be ruled out.The previously reported mechanisms of reduced glutamate uptake upon CaMKII inhibition in recombinant cell systems (Chawla et al., 2017;Underhill et al., 2015) may not be applicable to our study in intact brain slices.In addition, the cellular contribution of the perturbed glutamate uptake in the CaMKIIα KO slices needs to be explored further.In line with a reduced glutamate uptake capacity, mice lacking CaMKIIα have been found to display neuronal hyperexcitability translating into spontaneous epileptic seizures (Butler et al., 1995).Furthermore, knockdown of CaMKII in cultures of hippocampal neurons induces spontaneous recurrent discharges, indicative of epileptiform activity (Carter et al., 2006).Collectively, these studies suggest that loss of CaMKIIα function leads to a dysregulation of excitatory signaling, which in light of our study, may be mediated by impaired glutamate clearance.Intriguingly, mice lacking CaMKIIα also display larger infarct sizes after cerebral ischemia by middle cerebral artery occlusion (Waxham et al., 1996).The larger infarcts of CaMKIIα KO mice were recently confirmed in a photothrombotic stroke model, which also displayed marked hemorrhagic bleedings (Leurs et al., 2021).
Such worsened stroke outcome could be linked to impaired glutamate clearance, in turn causing excitotoxicity, being a major pathway of neurodegeneration in stroke (Lai et al., 2014).Pharmacological modulation of CaMKIIα was recently shown to reduce glutamate excitotoxicity in cultured neurons and reduce infarct volume in a stroke model (Griem-Krey et al., 2022;Leurs et al., 2021).How these beneficial outcomes may be related to cellular glutamate handling are subjects of future investigations.
It was recently revealed that CaMKIIα is the high-affinity binding site of the GABA metabolite γ-hydroxybutyrate (GHB) (Leurs et al., 2021).Intriguingly, intrahippocampal administration of low levels (0.1-0.5 μM) of GHB leads to prominent increases in extracellular glutamate levels, which could be normalized by co-infusion of a purported competitive inhibitor of high-affinity GHB-binding (Castelli et al., 2003;Ferraro et al., 2001).Regulation and pharmacology of CaMKIIα are highly complex processes and the authors did not reach a conclusive mechanism behind the observations, but GHB-CaMKIIα signaling is clearly linked to glutamate homeostasis.Given the compensation by other CaMKII isoforms in the CaMKIIα KO mouse, as discussed above, conclusions should be drawn with caution.However, our results, showing that loss of CaMKIIα does not disrupt the total brain glutamate amounts (Table 1), but reduces uptake and metabolism of exogenous glutamate, strengthen the link between CaMKIIα signaling and glutamatergic homeostasis.Finally, glutamate uptake is also closely coupled to cellular energy metabolism.Impaired glycolytic and mitochondrial function greatly reduce astrocyte glutamate uptake (Di Monte et al., 1999;Swanson et al., 1995).Furthermore, oxidative metabolism of glutamate can fuel its own uptake (McKenna, 2013) and inhibition of glutamate metabolism likewise reduces glutamate uptake in both astrocytes (Bauer et al., 2012) and isolated synaptosomes (Whitelaw & Robinson, 2013).Given the extensive reduction in [U-13 C]glucose metabolism in the CaMKIIα KO brain slices, it is conceivable that the reduced metabolic capacity could limit glutamate uptake and hereby cause the observed reduction in uptake of [U-

ACK N OWLED G M ENTS
Durita Poulsen is acknowledged for excellent technical support.

(
Figure 5) are presented as M + X, showing the direct and first turn metabolism of the substrates.The specific labeling patterns of [U-13 C] Pierce protein assay.The intracellular amounts of central amino acids were determined by HPLC analysis.Brain slice extracts of the [U-13 C]glutamate and [U-13 C]GABA incubations were also subjected to HPLC analysis to determine the slice uptake of glutamate and GABA, respectively.The tissue and slice extracts were reconstituted in water before reverse-phase HPLC analysis (Agilent Technologies, 1260 Infinity, Agilent ZORBAX Eclipse Plus C18 column) as described previously(Andersen, Westi, et al., 2021).Briefly, a pre-column derivatization with o-phthalaldehyde and fluorescent detection, λ ex = 338 nm, λ em = 390 nm, was performed.Gradient elution was performed with mobile phase A (10 mM NaH 2 PO 4 , 10 mM Na 2 B 4 O 7 , and 0.5 mM NaN 3 , pH 8.2) and mobile phase B (acetonitrile 45%: methanol 45%: H 2 O 10%, V:V:V).The amounts of amino acids were determined from analysis of standards containing the amino acids of interest.

F
Reduced oxidative glucose metabolism upon loss of CaMKIIα.Intracellular 13 C enrichment of TCA cycle intermediates and connected amino acids from cerebral cortical slices of wild-type control (black bars) and CaMKIIα knockout (KO) mice (blue bars) following [U-13 C] glucose metabolism.[U-13 C]Glucose was provided at a concentration of 5 mM.[U-13 C]Glucose enters the TCA cycle as acetyl CoA M + 2 units, resulting in 13 C enrichment in both TCA cycle intermediates and connected amino acids.The 13 C enrichment is presented as the molecular carbon labeling (MCL), reflecting the weighted average of 13 C accumulation in a metabolite.

2. 7 |
Experimental design and statistical analysesData are presented as mean ± standard error of the mean (SEM), with individual data points included.Each data point (depicted by a circle in the graphs) represents biological replicates (i.e., obtained from one individual animal), which is denoted by "n" in the figure legends.This exploratory study was not pre-registered.No randomization of animals or blinding of investigators were applied.Sample sizes were estimated based on previous similar experiments of two independent experimental groups with a predicted effect size (│μ 1 − μ 2 │/σ) of 2 (where μ 1 and μ 2 are the means of the experimental groups and σ is the standard deviation), with α = 0.05 and 80% power using the nQuery software.No exclusion criteria were pre-determined; however, one wild-type control sample from the [U-13 C]glutamate incubations (Figure4) was lost during GC-MS analysis.All statistical analyses were performed in Graph Pad Prism 9.No test for outliers was performed.Data were first tested for normality using the Shapiro-Wilk test (α = 0.05 13C isotope tracing in cerebral cortical slices of CaMKIIα KO and control mice.The brain slices were incubated in the presence of13 C enriched substrates with subsequent mass spectrometry analysis for13 C F I G U R E 4 CaMKIIα deletion leads to a reduction in glutamate uptake and subsequent metabolism.Intracellular 13 C enrichment of TCA cycle intermediates and connected amino acids from cerebral cortical slices of wild-type control (black bars) and CaMKIIα knockout (KO) mice (red bars) following [U-13 C]glutamate metabolism.[U-13 C]Glutamate was provided at a concentration of 0.2 mM in addition to 5 mM D-glucose.Uptake of [U-13 C]glutamate leads to M + 5 enrichment of the intracellular glutamate pool, hereby reflecting glutamate uptake.[U-13 C]Glutamate enters cellular metabolism as α-ketoglutarate M + 5. Direct metabolism of [U-13 C]glutamate leads M + 5/M + 4 13 C enrichments of connected metabolites, whereas first metabolism gives rise to M + 3/M + 2 13 C labeling.[U-13 C]Glutamate can also be converted directly into glutamine M + 5 by glutamine synthetase (GS) or GABA M + 4 by glutamate decarboxylase (GAD).AAT, aspartate aminotransferase; GDH, glutamate dehydrogenase.Full statistical report can be found in Supplementary File 2. Mean ± SEM, n = 5-6 from individual animals, Welch's two-tailed t-test or Mann-Whitney test, both with Benjamini-Hochberg correction.enrichment in cellular metabolites.First, we investigated the metabolism of glucose being the primary energy substrate of the brain (Figures1 and 2).Glucose is metabolized through the multi-step pathway glycolysis into pyruvate, which is in equilibrium with alanine and lactate via alanine aminotransferase (ALAT) and lactate dehydrogenase (LDH) activity, respectively.Alanine and lactate can hereby serve as surrogate markers of glycolytic activity.Metabolism of [U-13 C]glucose gives rise to pyruvate M + 3, which can be converted into either alanine M + 3 or lactate M + 3.No differences were found in the intracellular enrichment of alanine M + 3 (p = 0.94) or lactate M + 3 (p = 0.12) in cerebral cortical slices of CaMKIIα KO mice (Figure1).Brain slices release large quantities of lactate to the media during incubation(McNair et al., 2017).In accordance with the unchanged 13 C enrichment of the intracellular lactate pool, no differences were observed in the amounts of released lactate from the F I G U R E 5 Sustained GABA uptake and metabolism in CaMKIIα knockout mice.Intracellular 13 C enrichment of TCA cycle intermediates and connected amino acids from cerebral cortical slices of wild-type control (black bars) and CaMKIIα knockout (KO) mice (orange bars) following [U-13 C]GABA metabolism.[U-13 C]GABA was provided at a concentration of 0.2 mM in addition to 5 mM D-glucose.Uptake of [U-13 C]GABA leads to M + 4 enrichment of the intracellular GABA pool, hereby reflecting GABA uptake.[U-13 C]GABA enters cellular metabolism as succinate M + 4. Direct metabolism of [U-13 C]GABA leads M + 4/M + 3 13 C enrichments of connected metabolites, whereas first metabolism gives rise to M + 2 13 C labeling.Note that 50% of the 13 C labeling in α-ketoglutarate, glutamate, and glutamine from first turn metabolism of [U-13 C]GABA will be M + 1 (see Supplementary File 1).AAT, aspartate aminotransferase; GDH, glutamate dehydrogenase; GABA-T, GABA transaminase; GS, glutamine synthetase; SSADH, succinic semialdehyde dehydrogenase.Full statistical report can be found in Supplementary File 2. Mean ± SEM, n = 6 from individual animals, Welch's two-tailed t-test or Mann-Whitney test, both with Benjamini-Hochberg correction.
the mitochondria, pyruvate is converted into acetyl CoA by activity of the pyruvate dehydrogenase (PDH) complex.Conversion of pyruvate M + 3 from metabolism of [U-13 C]glucose generates acetyl CoA M + 2, which enters the TCA cycle and leads to 13 C enrichment of TCA cycle intermediates and connected amino acids (Figure2).The overall activity of oxidative glucose metabolism can be illustrated by the molecular carbon labeling (MCL), which the weighted average of 13 C labeling of a given metabolite.In the cerebral cortical slices of CaMKIIα KO mice, we observed reduced MCLs in the TCA cycle intermediates citrate (−12.8%,p = 0.016), α-ketoglutarate (−27.2%,p = 0.018), fumarate (−25.4%,p = 0.003), and malate (−27.7%,p = 0.004) from metabolism of [U-13 C]glucose (Figure2).Decreased MCLs of the two amino acids aspartate (−26.1%,p = 0.002) and glutamate (−18.5%,p = 0.005) were likewise observed.Since both aspartate and glutamate are linked to the TCA cycle, the reduced 13 C enrichment of these amino acids is likely a reflection of the overall reduced oxidative glucose metabolism.Glutamate also serves as the precursor for both glutamine and GABA synthesis, taking place in astrocytes and neurons, respectively.The MCLs were likewise reduced for glutamine (−12.9%,p = 0.020) and GABA (−20.1%,p = 0.001) from metabolism of [U-13 C]glucose in the CaMKIIα cerebral cortex.Collectively, these results demonstrate a prominent reduction in the rate of oxidative glucose metabolism in the cerebral cortex upon loss of CaMKIIα.
or glutamate (Figure6h) were present.Collectively, these results demonstrate a maintained synaptic mitochondrial function and glycolytic activity during non-stimulated resting conditions in the CaMKIIα KO mice.However, the capacity of both mitochondrial respiration and glycolysis becomes insufficient upon stimulation across several substrate combinations and points toward an overall lower synaptic metabolic function upon loss of CaMKIIα.
dative glucose metabolism may reflect a lower synaptic activity in the CaMKIIα KO brain.The magnitude of the reduced [U-13 C]glucose metabolism (21.3% average reduction in MCL) suggests that CaMKIIα function and synaptic plasticity claim a large fraction of the cerebral energy expenditure.However, the underlying mechanisms of the observed metabolic effects in the CaMKIIα KO mice are likely multifaceted and complex.Deletion of CaMKIIα may mediate acute effects by altered protein phosphorylation, but changes in transcriptional activity and post-natal neurodevelopmental effects may also contribute.In skeletal muscle, CaMKII signaling has been F I G U R E 6 Synaptic energetics are impaired upon CaMKIIα deletion.(a) Overview of synaptic energetics.Synaptosomes are isolated nerve endings displaying active glycolytic and mitochondrial activity.The synaptosomes were provided with different metabolic substrates: glucose alone (5 mM, blue bars, c & f), glucose in combination with pyruvate (both 5 mM, purple bars, d & g), or glucose in combination with glutamate (5 and 0.2 mM, respectively, red bars, e & h).The oxygen consumption rate (OCR) reflecting mitochondrial respiration and the extracellular acidification rate (ECAR) reflecting glycolytic activity were determined.(b) Overview of assay.Representative trace of OCR of isolated synaptosomes provided with glucose in combination with pyruvate.Two baseline measurements were followed by sequential application of the following compounds: (1) the neurotoxin veratridine preventing closure of voltage-gated sodium channel leading to a substantial sodium influx, (2) the ATP synthase inhibitor oligomycin A, (3) the mitochondrial uncoupler FCCP leading to maximal respiration, (4) the inhibitors of the electron transport chain rotenone and antimycin A. Four OCR parameters were derived from the obtained respiratory profiles: basal respiration, sodium channel activation, proton leak, and uncoupled respiration.The non-mitochondrial OCR was subtracted all OCR values prior to analyses.(c-e) OCR of isolated synaptosomes of wild-type control (black bars) and CaMKIIα knockout (KO) mice (colored bars depending on substrate).(f-h) ECAR of isolated synaptosomes of wild-type control (black bars) and CaMKIIα KO mice (colored bars depending on substrate).Full statistical report can be found in Supplementary File 2. Mean ± SEM, n = 6 from individual animals, paired t-test or Wilcoxon matched-pairs rank test, both with Benjamini-Hochberg correction.
pensate if activity of one isoform is lost, which may have affected the results of this study.Compensatory effects of other CaMKs, and connected pathways, are likely extensive upon CaMKIIα deletion and could lead to an underestimation of the metabolic component of CaMKIIα signaling.Elucidating the full compensatory profile of CaMKIIα deletion is beyond the scope of this report, but further exploration of the proposed CaMKII-AMPK-FOXO3 signaling pathway in the brain may aid to further reveal the metabolic significance of cerebral CaMKIIα signaling.
, suggesting that deletion of CaMKIIα may cause reduced mitochondrial function via altered FOXO3 signaling.Our observations of hampered mitochondrial activity during stimulation could be mediated by the mechanisms outlined above and underlines that CaMKIIα function plays a functional role in mitochondrial respiration in the brain.
13 C]glutamate.Collectively, our results link CaMKIIα function to brain energy metabolism and neurotransmitter homeostasis.In particular, neuronal energetics and glutamate homeostasis are dependent on CaMKIIα activity, implicating a significant metabolic role of CaMKIIα in the brain.AUTH O R CO NTR I B UTI O N S Jens V. Andersen performed slice incubations and Seahorse experiments.Emil W. Westi assisted with sample analysis.All authors interpreted the data and provided critical input to the final manuscript.Jens V. Andersen and Petrine Wellendorph conceived the overall project and Jens V. Andersen wrote the original manuscript.