Magnesium efflux from Drosophila Kenyon cells is critical for normal and diet-enhanced long-term memory

The proverbial saying ‘you are what you eat’ perfectly summarizes the concept that our diet can influence both our mental and physical health. We know that foods that are good for the heart, such as nuts, oily fish and berries, are also good for the brain. We know too that vitamins and minerals are essential for overall good health. But is there any evidence that increasing your intake of specific vitamins or minerals could help boost your brain power?

While it might sound almost too good to be true, there is some evidence that this is the case for at least one mineral, magnesium. Studies in rodents have shown that adding magnesium supplements to food improves how well the animals perform on memory tasks. Both young and old animals benefit from additional magnesium. Even elderly rodents with a condition similar to Alzheimer’s disease show less memory loss when given magnesium supplements. But what about other species?

Wu et al. now show that magnesium supplements also boost memory performance in fruit flies. One group of flies was fed with standard cornmeal for several days, while the other group received cornmeal supplemented with magnesium. Both groups were then trained to associate an odor with a food reward. Flies that had received the extra magnesium showed better memory for the odor when tested 24 hours after training.

Wu et al. show that magnesium improves memory in the flies via a different mechanism to that reported previously for rodents. In rodents, magnesium increased levels of a receptor protein for a brain chemical called glutamate. In fruit flies, by contrast, the memory boost depended on a protein that transports magnesium out of neurons. Mutant flies that lacked this transporter showed memory impairments. Unlike normal flies, those without the transporter showed no memory improvement after eating magnesium-enriched food. The results suggest that the transporter may help adjust magnesium levels inside brain cells in response to neural activity.

Humans produce four variants of this magnesium transporter, each encoded by a different gene. One of these transporters has already been implicated in brain development. The findings of Wu et al. suggest that the transporters may also act in the adult brain to influence cognition. Further studies are needed to test whether targeting the magnesium transporter could ultimately hold promise for treating memory impairments.

Magnesium (Mg²⁺) plays a critical role in cellular metabolism and is considered to be an essential co-factor for more than 350 enzymes (Romani and Scarpa, 2000; Vink and Nechifor, 2011). As a result, alterations of Mg²⁺ homeostasis are associated with a broad range of clinical conditions, including those affecting the nervous system, such as glaucoma (DeToma et al., 2014), Parkinson’s disease (Hermosura et al., 2005; Hermosura and Garruto, 2007; Lin et al., 2014; Shindo et al., 2016), Alzheimer’s disease (Andrási et al., 2000; Andrási et al., 2005; Cilliler et al., 2007; Durlach et al., 1997; Glick, 1990; Lemke, 1995; Chui et al., 2011; Vural et al., 2010), anxiety (Sartori et al., 2012), depression (Whittle et al., 2011; Murck, 2002; Murck, 2013; Rasmussen et al., 1990; Ghafari et al., 2015), and intellectual disability (Arjona et al., 2014).

Perhaps surprisingly, increasing brain Mg²⁺ through diet can enhance neuronal plasticity and memory performance of young and aged rodents, measured in a variety of behavioral tasks (Slutsky et al., 2010; Landfield and Morgan, 1984; Mickley et al., 2013; Abumaria et al., 2013). In addition, elevated Mg²⁺ reduced cognitive deficits in a mouse model of Alzheimer’s disease (Li et al., 2013) and enhanced the extinction of fear memories (Abumaria et al., 2011). These apparently beneficial effects have led to the proposal that dietary Mg²⁺ may have therapeutic value for patients with a variety of memory-related problems (Billard, 2011).

Despite the large number of potential sites of Mg²⁺ action in the brain, the memory-enhancing property in rodents has largely been attributed to increases in hippocampal synaptic density and the activity of N-methyl-D-aspartate glutamate receptors (NMDARs). Extracellular Mg²⁺ blocks the channel pore of the NMDAR and thereby inhibits the passage of other ions (Mayer et al., 1984; Bekkers and Stevens, 1993; Jahr and Stevens, 1990; Nowak et al., 1984). Importantly, prior neuronal depolarization, driven by other transmitter receptors, is required to release the Mg²⁺ block on the NMDAR and permit glutamate-gated Ca²⁺ influx. The NMDAR therefore plays an important role in neuronal plasticity as a potential Hebbian coincidence detector. Acute elevation of extracellular Mg²⁺ concentration ([Mg²⁺]_e) within the physiological range (0.8–1.2 mM) can antagonize induction of NMDAR-dependent long-term potentiation (Dunwiddie and Lynch, 1979; Malenka et al., 1992; Malenka and Nicoll, 1993; Slutsky et al., 2004). In contrast, increasing [Mg²⁺]_e for several hours in neuronal cultures leads to enhancement of NMDAR mediated currents and facilitation of the expression of LTP (Slutsky et al., 2004). The enhancing effects of increased [Mg²⁺]_e were also observed in vivo in the brain of rats fed with Mg²⁺-L-threonate (Slutsky et al., 2010). Hippocampal neuronal circuits undergo homeostatic plasticity (Turrigiano, 2008) to accommodate the increased [Mg²⁺]_e by upregulating expression of NR2B subunit containing NMDARs (Slutsky et al., 2004; Slutsky et al., 2010). The higher density of hippocampal synapses with NR2B containing NMDARs are believed to compensate for the chronic increase in [Mg²⁺]_e by enhancing NMDAR currents during burst firing. In support of this model, mice that are genetically engineered to overexpress NR2B exhibit enhanced hippocampal LTP and behavioral memory (Tang et al., 1999).

Olfactory memory in Drosophila involves a heterosynaptic mechanism driven by reinforcing dopaminergic neurons, which results in presynaptic depression of cholinergic connections between odor-activated mushroom body (MB) Kenyon cells (KCs) and downstream mushroom body output neurons (MBONs) (Schwaerzel et al., 2003; Aso et al., 2010; Aso et al., 2012; Claridge-Chang et al., 2009; Burke et al., 2012; Liu et al., 2012; Plaçais et al., 2013; Owald et al., 2015; Hige et al., 2015; Barnstedt et al., 2016; Perisse et al., 2016; Aso et al., 2014; Owald and Waddell, 2015). In addition, olfactory information is conveyed to KCs by cholinergic transmission from olfactory projection neurons (Yasuyama et al., 2002; Leiss et al., 2009). Although it is conceivable that glutamate is delivered to the MB network via an as yet to be identified route, there is currently no obvious location for NMDAR-dependent plasticity in the known architecture of the cholinergic input or output layers (Barnstedt et al., 2016). The fly therefore provides a potential model to investigate other mechanisms through which dietary Mg²⁺ might enhance memory.

The reinforcing effects of dopamine depend on the Dop1R D1-type dopamine receptor (Kim et al., 2007; Qin et al., 2012; Handler et al., 2019), which is positively coupled with cAMP production (Tomchik and Davis, 2009; Boto et al., 2014). Moreover, early studies in Drosophila identified the dunce and rutabaga encoded cAMP phosphodiesterase and type I Ca^2+-stimulated adenylate cyclase, respectively, to be essential for olfactory memory (Dudai et al., 1976; Byers et al., 1981; Dudai and Zvi, 1984; Chen et al., 1986; Livingstone et al., 1984; Levin et al., 1992). Studies in mammalian cells have shown that hormones or agents that increase cellular cAMP level often elicit a significant Na⁺-dependent extrusion of Mg²⁺ into the extracellular space (Romani and Scarpa, 1990b; Romani and Scarpa, 1990a; Romani and Scarpa, 2000; Vink and Nechifor, 2011; Vormann and Günther, 1987). However, it is unclear whether Mg²⁺ extrusion plays any role in memory processing.

Here we demonstrate that Drosophila long-term memory (LTM) can be enhanced with dietary Mg²⁺ supplementation. We find that the unextended (uex) (Maeda, 1984; Coulthard et al., 2010) gene, which encodes a functional fly ortholog of the mammalian Cyclin M2 Mg²⁺-efflux transporter (CNNM) proteins, is critical for the memory enhancing property of Mg²⁺. UEX function in MB KCs is required for LTM and functional restoration of uex reveals the MB to be the key site of Mg²⁺-dependent memory enhancement. Chronically changing cAMP metabolism by introducing mutations in the dnc or rut genes alters the cellular localization of UEX. Moreover, mutating the conserved cyclic nucleotide-binding homology (CNBH) domain in UEX uncouples an essential role for uex from its function in memory. UEX-driven Mg²⁺ efflux is required for slow rhythmic maintenance of KC Mg²⁺ levels suggesting a potential role for Mg²⁺ flux in memory processing.

We observed an enhancement of olfactory LTM performance when flies were fed for 4 days before training with food supplemented with 80 mM [Mg²⁺]. This result resembles that reported in rats, although longer periods of feeding were required to raise brain [Mg²⁺] to memory-enhancing levels (Slutsky et al., 2010). A difference in optimal feeding time may reflect the size of the animal and perhaps the greater bioavailability of dietary Mg²⁺ in Drosophila. Whereas Mg²⁺-L-threonate (MgT) was a more effective means of delivering Mg²⁺ than magnesium chloride in rats (Slutsky et al., 2010), we observed a similar enhancement of memory performance when flies were fed with magnesium chloride, magnesium sulfate, or MgT (data not shown).

Elevating [Mg²⁺]_e in the rat brain leads to a compensatory upregulation of expression of the NR2B subunit of the NMDAR and therefore an increase in the proportion of postsynaptic NR2B-containing NMDARs. This class of NMDARs have a longer opening time (Chen et al., 1999; Erreger et al., 2005) suggesting that this switch in subunit composition represents a homeostatic plasticity mechanism (Turrigiano, 2008) to accommodate for the increased NMDAR block imposed by increasing [Mg²⁺]_e. Moreover, overexpression of NR2B in the mouse forebrain can enhance synaptic facilitation and learning and memory performance (Tang et al., 1999), supporting an increase in NR2B being an important factor in Mg²⁺-enhanced memory. However, even in the original in vitro study of Mg²⁺-enhanced synaptic plasticity (Slutsky et al., 2004), it was noted that NMDAR currents were insufficient to fully explain the observed changes.

Our NMDAR subunit loss-of-function studies in the Drosophila KCs did not impair regular or Mg²⁺-enhanced memory. Furthermore, we did not detect an obvious change in the levels of brain-wide expression of glutamate receptor subunits in Mg²⁺-fed flies. Although NMDAR activity has previously been implicated in Drosophila olfactory memory, the effects were mostly ascribed to function outside the MB (Xia et al., 2005; Wu et al., 2007). In addition, overexpressing Nmdar1 in all neurons, or specifically in all KCs, did not alter STM or LTM. Ectopic overexpression in the MB of an NMDAR^N631Q version, which cannot be blocked by Mg²⁺, impaired LTM (Miyashita et al., 2012). However, this mutation permits ligand-gated Ca²⁺ entry, without the need for correlated neuronal depolarization, which may perturb KC function in unexpected ways. It is perhaps most noteworthy that learning-relevant synaptic depression in the MB can be driven by dopaminergic teaching signals delivered to cholinergic output synapses from odor-responsive KCs to specific MBONs (Claridge-Chang et al., 2009; Aso et al., 2012; Burke et al., 2012; Liu et al., 2012; Owald et al., 2015; Hige et al., 2015; Barnstedt et al., 2016; Perisse et al., 2016; Aso et al., 2014; Owald and Waddell, 2015; Handler et al., 2019). It is conceivable that KCs receive glutamate, from a source yet to be identified, but there is currently no obvious place in the MB network for NMDAR-dependent plasticity. Evidence therefore suggests that normal and Mg²⁺-enhanced Drosophila LTM is independent of NMDAR signaling in KCs. In addition, our MagFRET measurements indicate that Mg²⁺ feeding also increases the [Mg²⁺]_i of αβ KCs by approximately 50 µM.

We identified a role for uex, the single fly ortholog of the evolutionarily conserved family of CNNM-type Mg²⁺ efflux transporters (Ishii et al., 2016). There are four distinct CNNM genes in mice and humans, five in C. elegans, and two in zebrafish (Ishii et al., 2016; Arjona et al., 2013). The uex locus produces four alternatively spliced mRNA transcripts, but all encode the same 834 aa protein. The precise role of CNNM proteins in Mg²⁺ transport is somewhat contentious (Funato et al., 2018a; Arjona and de Baaij, 2018; Funato et al., 2018b; Giménez-Mascarell et al., 2019). Some propose that CNNM proteins are direct Mg²⁺ transporters, whereas others favor that they function as sensors of intracellular Mg²⁺ concentration [Mg²⁺]_i and/or regulators of other Mg²⁺ transporters. We found that ectopic expression of Drosophila UEX enhances Mg²⁺ efflux in HEK293 cells and that endogenous UEX limits [Mg²⁺]_i in αβ KCs in the fly brain. Therefore, if UEX is not itself a Mg²⁺ transporter, it must be able to interact effectively with human Mg²⁺ efflux transporters and to influence Mg²⁺ extrusion in Drosophila. Since UEX is the only CNNM protein in the fly, it may serve all the roles of the four individual mammalian CNNMs. However, the ability of mouse CNNM2 to restore memory capacity to uex mutant flies suggests that the memory-relevant UEX function can be substituted by that of CNNM2.

Interestingly, none of the disease-relevant variants of CNNM2 were able to complement the memory defect of uex mutant flies. The CNNM2 T568I variant substitutes a single amino acid in the second CBS domain (Arjona et al., 2014). The oncogenic protein tyrosine phosphatases of the PRL (phosphatase of regenerating liver) family bind to the CBS domains of CNNM2 and CNNM3 and can inhibit their Mg²⁺ transport function (Hardy et al., 2015; Giménez-Mascarell et al., 2017; Zhang et al., 2017). It will therefore be of interest to test the role of the UEX CBS domains and whether fly PRL-1 regulates UEX activity.

RNA-seq analysis reveals that uex is strongly expressed in the larval and adult fly digestive tract and nervous systems, as well as the ovaries (Gelbart and Emmert, 2010; Croset et al., 2018; Davie et al., 2018) suggesting that many uex mutations will be pleiotropic. Our uexΔ allele, which deletes 272 amino acids (including part of the second CBS and the entire CNBH domain) from the UEX C-terminus, results in developmental lethality when homozygous, demonstrating that uex is an essential gene. Mammalian CNNM4 is localized to the basolateral membrane of intestinal epithelial cells (Yamazaki et al., 2013). There it is believed to function in transcellular Mg²⁺ transport by exchanging intracellular Mg²⁺ for extracellular Na⁺ following apical entry through TRPM7 channels. Lethality in Drosophila could therefore arise from an inability to absorb sufficient Mg²⁺ through the larval gut. However, neuronally restricted expression of uex^RNAiwith elav-GAL4 also results in larval lethality (data not shown), suggesting UEX has an additional role in early development of the nervous system, like CNNM2 in humans and zebrafish (Arjona et al., 2014; Accogli et al., 2019). Perhaps surprisingly, flies carrying homozygous or trans-heterozygous combinations of several hypomorphic uex alleles have defective appetitive and aversive memory performance, yet they seem otherwise unaffected.

Genetically engineering the uex locus to add a C-terminal HA tag to the UEX protein allowed us to localize its expression in the brain. Labeling is particularly prominent in all major classes of KCs. Restricting knockdown of uex expression to all αβ KCs of adult flies, or even just the αβ_c subset reproduced the LTM defect. The LTM impairment was evident if uex^RNAi expression in αβ neurons was restricted to adult flies, suggesting UEX has a more sustained role in neuronal physiology. In contrast, knocking down uex expression in either the αβ_s or α′β′ neurons did not impair LTM. Activity of α′β′ neurons is required after training to consolidate appetitive LTM (Krashes and Waddell, 2008), whereas αβ_c and αβ_s KC output, together and separately, is required for its expression (Krashes and Waddell, 2008; Perisse et al., 2013). Therefore, observing normal LTM performance in flies with uex loss-of-function in αβ_s and α′β′ neurons argues against a general deficiency of αβ neuronal function when manipulating uex.

Dietary Mg²⁺ could not enhance the defective LTM performance of flies that were constitutively uex mutant, or harbored αβ KC-restricted uex loss-of-function. However, expressing uex in the αβ KCs of uex mutant flies restored the ability of Mg²⁺ to enhance performance. Therefore, the αβ KCs are the cellular locus for Mg^2+-enhanced memory in the fly.

It perhaps seems counterintuitive that UEX-directed magnesium efflux is required in KCs to support the memory-enhancing effects of Mg²⁺ feeding, when dietary Mg²⁺ elevates KC [Mg²⁺]_i. At this stage, we can only speculate as to why this is the case. We assume that the brain and αβ KCs, in particular, have to adapt in a balanced way to the higher levels of intracellular and extracellular Mg²⁺ that result from dietary supplementation. Our live-imaging of KC [Mg²⁺]_i in wild-type and uex mutant brains suggests that UEX-directed efflux is likely to be an essential factor in the active, and perhaps stimulus-evoked, homeostatic maintenance of these elevated levels.

A number of mammalian cell-types extrude Mg²⁺ in a cAMP-dependent manner, a few minutes after being exposed to β-adrenergic stimulation (Romani and Scarpa, 2000; Vormann and Günther, 1987; Jakob et al., 1989; Romani and Scarpa, 1990b; Romani and Scarpa, 1990a; Vormann and Günther, 1987; Günther et al., 1990; Howarth et al., 1994). The presence of a CNBH domain suggests that UEX and CNNMs could be directly regulated by cAMP. We tested the importance of the CNBH by introducing an R622K amino acid substitution that should block cAMP binding in the UEX CNBH. This subtle mutation abolished the ability of the uex^R622K transgene to restore LTM performance to uex mutant flies. We also used CRISPR to mutate the CNBH in the native uex locus. Although deleting the CNBH from CNNM4 abolished Mg²⁺ efflux activity (Chen et al., 2018), flies homozygous for the uex^T626NRR lesion were viable, demonstrating that they retain a sufficient level of UEX function. However, these flies exhibited impaired immediate and long-term memory. In addition, the performance of uex^T626NRR flies could not be enhanced by Mg²⁺ feeding. These data demonstrate that an intact CNBH is a critical element of memory-relevant UEX function. Binding of clathrin adaptor proteins to the CNNM4 CNBH has been implicated in basolateral targeting (Hirata et al., 2014), suggesting that UEX^T626NRR might be inappropriately localized in KCs. Furthermore, KC expression of the CNNM2 E122K mutant variant, which retains residual function but has a trafficking defect (Arjona et al., 2014), did not restore the uex LTM defect.

Although it has been questioned whether the CNNM2/3 CNBH domains bind cyclic nucleotides (Chen et al., 2018), we found that FSK evoked an increase in αβ KC [Mg²⁺]_i that was sensitive to uex mutation, and that UEX::HA was mislocalized in rut²⁰⁸⁰ adenylate cyclase (Han et al., 1992) and dnc¹ phosphodiesterase (Dudai et al., 1976) learning defective mutant flies. Whereas UEX::HA label was evenly distributed in γ, αβ_c, and αβ_s KCs in wild-type flies, UEX::HA label was diminished in the γ and αβ_s KCs and was stronger in αβ_c neurons in rut²⁰⁸⁰ and dnc¹ mutants. The chronic manipulations of cAMP in the mutants are therefore consistent with cAMP impacting UEX localization, perhaps by interacting with the CNBH. In addition, altered UEX localization may contribute to the memory defects of rut²⁰⁸⁰ and dnc¹ flies.

Our physiological data using Magnesium Green in mammalian cell culture and the genetically encoded MagIC reporter in αβ KCs demonstrate that fly UEX facilitates Mg²⁺efflux. Stimulating the fly brain with FSK evoked a greater increase in αβ KC [Mg²⁺]_i in uex mutant brains than in wild-type controls which provides the first evidence that UEX limits a rise in [Mg²⁺]_i in Drosophila KCs. Our MagIC recordings also revealed a slow oscillation (centered around 0.015 Hz, approximately once a minute) of αβ KC [Mg²⁺]_i that was dependent on UEX. We do not yet understand the physiological function of this [Mg²⁺]_i fluctuation although it likely reflects a homeostatic systems-level property of the cells. Biochemical oscillatory activity plays a crucial role in many aspects of cellular physiology (Novák and Tyson, 2008). Most notably, circadian timed fluctuation of [Mg²⁺]_i links dynamic cellular energy metabolism to clock-controlled translation through the Mg²⁺ sensitive mTOR (mechanistic target of rapamycin) pathway (Feeney et al., 2016). It is therefore possible that slow Mg²⁺ oscillations could unite roles for cAMP, UEX, energy flux (Plaçais et al., 2017), and mTOR-dependent translation underlying LTM-relevant synaptic plasticity (Casadio et al., 1999; Huber et al., 2000; Beaumont et al., 2001; Hou and Klann, 2004; Hoeffer et al., 2008).

Behaviour data from T-maze assays are deposited in Dryad Digital Repository (https://doi.org/10.5061/dryad.q2bvq83hs). All other data generated or analysed during this study are included in the manuscript and supporting files.

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Acceptance summary:

Wu et al. discover elevated dietary magnesium enhances long-term memory in Drosophila. They then identify target neurons (α/β Kenyon Cells of the mushroom body) and a protein critical for the effect, the CNNM2 ortholog Unextended (UEX), which the authors implicate in magnesium efflux. The study is well-performed and conclusions justified by the data presented. Overall, this paper marks an important step in examining a highly significant, but understudied topic.

Decision letter after peer review:

Thank you for submitting your article "Magnesium efflux from Drosophila Kenyon Cells is critical for normal and diet-enhanced long-term memory" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by K VijayRaghavan as the Senior Editor and Reviewing Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

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Summary:

Supplemental dietary magnesium enhances learning and memory in rats, an observation with implications for humans. The underlying mechanisms are not known, although it is correlated with increased hippocampal synaptic transmission and plasticity. Here Wu et al. discover elevated dietary magnesium also enhances long term memory in Drosophila. They then identify target neurons (α/β Kenyon Cells of the mushroom body) and a protein critical for the effect, the CNNM2 ortholog Unextended (UEX), which the authors implicate in magnesium efflux. The study is well-performed and conclusions justified by the data presented. Overall, this paper marks an important step in examining a highly significant, but understudied topic. How dietary magnesium influences memory is at least in part, is ascribed to changes in synaptic density via elevated NMDA receptor expression. However, this connection cannot explain all facets and, given the importance of memory-enhancing treatments for sufferers of neurodegenerative disease, it is an important question. The authors discover that elevated dietary magnesium enhances long term memory performance in Drosophila. They identify a likely set of target neurons (α/β Kenyon Cells) and a protein critical for this effect, a multi-pass transmembrane protein, the Drosophila CNNM2 ortholog Unextended (UEX). The authors demonstrate that UEX acts in the α/β KCs of the mushroom body to support both appetitive and aversive long term (24h) memory performance, and provide evidence that UEX promotes magnesium efflux. UEX functions in the αβ Kenyon Cells in the mushroom body and requires its cyclic nucleotide binding domain for proper function. Consistently, in spirit, alterations of cAMP levels using classical memory mutants alter localization of UEX. Finally, the authors identify a slow oscillation of magnesium in Kenyon Cells that requires UEX function.

At a technical level, the behavioural and molecular genetic aspects of the study (including the tagging of the endogenous protein and the creation of site-specific genomic mutants) are very well done. These experiments are thorough and well-executed, with appropriate controls, and the conclusions drawn justified by the data presented.

There are many issues raised by the study that provide fertile topics for future work, from details such as how cyclic nucleotide binding affects UEX trafficking to bigger picture issues including the mechanism by which UEX-mediated magnesium efflux contributes to memory and the dietary effects of magnesium on memory, not to mention what are the key targets of magnesium and how magnesium oscillations contribute to memory. However, while those are worthy topics for future studies, the paper as it stands constitutes a significant advance worthy of publication in eLife.

However, there are several concerns that need to be addressed before acceptance. We would prefer that they be addressed experimentally, where indicated, and done so speedily. We understand that the absence of reagents, time and access could make this a difficult or impossible requirement to meet. In that case, the manuscript should insert appropriate caveats in interpretation.

Revisions for this paper:

1) The increase in α/β lobe MagFRET-1 FRET signal in Figure 1E is modestly enhanced by 80 mM dietary MgCl₂ (under 10%). Given the apparent affinity of the sensor for Mg²⁺ (Km ~ 150 microM ) and the ~50% increase in FRET signal upon Mg²⁺ binding, do the authors have a sense of what such an increase would correspond to in terms of actual Mg²⁺ concentration?

2) On a related point, the MagFRET-1 experiment was done on formaldehyde fixed tissue. Is it clear that the sensor provides an accurate reflection of in vivo magnesium levels after fixation and washing?

3) Uex mutations affect both aversive and appetitive memory. Is the same true for Mg²⁺ supplementation?

4) How do the authors rationalize the role of a magnesium efflux transporter (UEX) in supporting the effect of elevated dietary magnesium? Naively, one would anticipate that UEX would act to counter the effects of higher levels of magnesium.

5) In a few places, the jargon and common practice of Drosophila genetics is not well enough explained for a non-fly audience. For example, the discussion of Nmdar RNAi (subsection “Mg²⁺ enhanced memory is independent of NMDAR in the mushroom bodies”) and the use of GAL80-TS (subsection “A role for uex in the mushroom bodies”) should be more generally described to understand the experimental logic.

6) The authors indicate that αβc knockdown causes a phenotype (Figure 3C) but UAS expression in those cells does not rescue the phenotype (Figure 4A). Do the authors have an explanation for the discrepancy?

Revisions potentially involving experimental additions:

7) In Figure 5, the human relative CNNM2 is used to rescue the uex mutant fly. Mutant CNNM2 variants associated with disease in humans are reported to not rescue the fly, but it is not clear whether the failure to rescue is caused by altered protein function or a failure of protein expression/localization. Expression and localization of the CNNM2 mutant proteins should be examined. If that is not possible, the authors should note these as possible explanations for the failure to rescue.

Also, what do these point mutants do? Can the authors do cell assays to verify that magnesium efflux is blocked in these mutants (as in Figure 5—figure supplement 2)? Or alternatively indicate references and data where appropriate

8) The impact of the T626NRR mutation on protein function is unclear, raising the concern that it could have a broader impact on the protein than simply altering nucleotide binding domain function. Is the mutant protein expressed and localized properly? Does the mutant protein have altered cAMP binding? Does it affect magnesium efflux activity? Barring massive experiments, a repeat of their MagIC experiment from Figure 8 using that specific construct would be satisfactory. If the authors do not know what the biochemical impact of this lesion is and are unable to carry out this experiment at present, they should insert appropriate caveats when interpreting the phenotype of this mutant.

9) Why were the HEK cell experiments done with a human CNNM4 while the fly experiments were done with human CNNM2? They're homologous, but why not use the same protein in each experiment to strengthen the parallels.

Revisions for this paper:

1) The increase in α/β lobe MagFRET-1 FRET signal in Figure 1E is modestly enhanced by 80 mM dietary MgCl₂ (under 10%). Given the apparent affinity of the sensor for Mg²⁺ (Km ~ 150 microM ) and the ~50% increase in FRET signal upon Mg²⁺ binding, do the authors have a sense of what such an increase would correspond to in terms of actual Mg²⁺ concentration?

As suggested by the reviewer, we have now used the MagFRET-1 signal enhancement observed in the α/β lobes to estimate the change in Mg²⁺ concentration that results from 80mM dietary MgCl₂.

If full occupancy of the reporter gives a 50% increase in FRET and the Kd (concentration that produces half occupancy) = 148uM then a 50% increase in FRET corresponds to a real change in [Mg²⁺] of 296µM.

In Figure 1E, for the α lobe the mean change in the emission ratio as a percentage is = (0.8340-0.7919)/ 0.7919 = 5.32% which corresponds to Δ[Mg²⁺] of 31.49 µM. For the β Lobe, the mean change in emission ratio as a percentage is = (0.7356-0.6650)/ 0.6650 = 10.60%, corresponding to Δ[Mg²⁺] of 62.75 µM. Averaging across the α and β lobes therefore gives us a Δ[Mg²⁺] 47.12 µM. We therefore conclude that feeding the flies 80mM of dietary MgCl₂ produces an ~50 µM increase of KC [Mg²⁺]_i on average.

The following sentence has been added:

“Given the affinity of MagFRET-1 (Kd = 148 µM) and the ~50% increase in FRET signal upon Mg²⁺ binding (Lindenburg et al., 2013), we estimate that the ~8% enhancement of the MagFRET signal measured in flies fed 80mN MgCl₂ corresponds to an ~50 µM increase of αβ KC [Mg²⁺]i on average.”

2) On a related point, the MagFRET-1 experiment was done on formaldehyde fixed tissue. Is it clear that the sensor provides an accurate reflection of in vivo magnesium levels after fixation and washing?

We do not know how fixation and washing might change the property of the sensor. However, we think it’s reasonable to assume that any change that occurs from fixation and/or washing change would happen in a consistent way in the brains from normal flies and those fed with high dietary Mg²⁺. Therefore, the difference in [Mg²⁺]_i observed between the conditions is presumably retained after the fixation and washing process, that is equally applied to all flies.

We did also try to use MagFRET and MagIC to compare the [Mg²⁺]_i in normal and high Mg²⁺ diet flies using an in vivo measurement. However, the variation in these in vivo experiments appears to be too high to draw any convincing conclusions. We provide the MagIC data in Author response image 1 for the reviewers but do not include it in the manuscript:

Author response image 1

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Following these in vivo experiments with MagFRET and MagIC we consider it possible that the fixation procedure applied to MagFRET may actually help to capture the current brain state and thereby enable a quantitative comparison between conditions. Perhaps this is especially important when the change in concentration is as subtle as ~50 µM.

3) Uex mutations affect both aversive and appetitive memory. Is the same true for Mg++ supplementation?

Yes, the same is also true for Mg²⁺ supplementation. These new data have been added to the manuscript as Figure 4—figure supplement 1. The corresponding citations in the manuscript have been updated.

The following text has been added:

“We also tested whether 4 days of 80mM MgCl₂ supplementation enhanced 24 hr memory performance following aversive spaced training. Again, memory of wild-type, but not uex^MI01943 mutant flies showed enhancement (Figure 4—figure supplement 1).”

4) How do the authors rationalize the role of a magnesium efflux transporter (UEX) in supporting the effect of elevated dietary magnesium? Naively, one would anticipate that UEX would act to counter the effects of higher levels of magnesium.

We can only provide a hypothetical suggestion at the moment and we have added the following statement in the Discussion:

“It perhaps seems counterintuitive that UEX-directed magnesium efflux is required in KCs to support the memory-enhancing effects of Mg²⁺ feeding, when dietary Mg²⁺ elevates KC [Mg²⁺]_i. […] Our live-imaging of KC [Mg²⁺]_i in wild-type and uex mutant brains suggests that UEX-directed efflux is likely to be an essential factor in the active, and perhaps stimulus evoked, homeostatic maintenance of these elevated levels.”

5) In a few places, the jargon and common practice of Drosophila genetics is not well enough explained for a non-fly audience. For example, the discussion of Nmdar RNAi (subsection “Mg²⁺ enhanced memory is independent of NMDAR in the mushroom bodies”) and the use of GAL80-TS (subsection “A role for uex in the mushroom bodies”) should be more generally described to understand the experimental logic.

On re-reading the manuscript we now see the overly heavy jargon and extent of assumptions of knowledge. We have amended the section to more clearly describe the reagents used for RNA interference (RNAi) targeting the NMDAR genes in the fly. We have also improved the section in the text related to the logic and use of GAL80-TS.

“We next directly tested whether Mg²⁺ enhanced memory required NMDAR function, by knocking down expression of the Nmdar1 or Nmdar2 genes using transgenic UAS-driven RNA interference (RNAi) constructs. […] In contrast, more selective expression of this UAS-Nmdar1^RNAi in long-term memory (LTM) relevant αβ KCs using c739-GAL4 did not significantly impair 24 hr memory performance (Figure 1—figure supplement 1B).”

“To reduce the likelihood that the uex^RNAi associated memory defect results from a developmental consequence, we also restricted UAS-uex^RNAi expression to adulthood using GAL80^ts-mediated temporal control (McGuire et al., 2003). […] Restricting UAS-uex^RNAi expression to αβ KCs in adult flies using c739-GAL4 with GAL80^ts produced a similar 24 hr specific memory defect to that observed when UAS-uex^RNAi was expressed without temporal control (Figure 3D-F).”

6) The authors indicate that αβc knockdown causes a phenotype (Figure 3C) but UAS expression in those cells does not rescue the phenotype (Figure 4A). Do the authors have an explanation for the discrepancy?

To clarify the interpretation of results, we added the following:

“Finding that αβ_c RNAi knockdown of uex produces a memory defect (Figure 3C) but UAS-uex expression in αβ_c does not rescue the uex^MI01943 mutant defect (Figure 4A) suggests that UEX function in αβ_c KCs is essential for appetitive LTM, whereas both the αβ_c and αβ_s KCs need to have functional UEX to support LTM. “

Revisions potentially involving experimental additions:

7) In Figure 5, the human relative CNNM2 is used to rescue the uex mutant fly. Mutant CNNM2 variants associated with disease in humans are reported to not rescue the fly, but it is not clear whether the failure to rescue is caused by altered protein function or a failure of protein expression/localization. Expression and localization of the CNNM2 mutant proteins should be examined. If that is not possible, the authors should note these as possible explanations for the failure to rescue.

Also, what do these point mutants do? Can the authors do cell assays to verify that magnesium efflux is blocked in these mutants (as in Figure 5—figure supplement 2)? Or alternatively indicate references and data where appropriate.

We have now confirmed the expression of the CNNM2 variants in the fly brain by immuno-staining using the C-terminal HA tag that is added to all of the CNNM2 variant cDNA clones. These data show that every variant is expressed at comparable levels when driven by c739-GAL4. These data have been added as a new Figure 5—figure supplement 1.

Regarding the effect of these CNNM2 mutant variants on magnesium transporting, experiments were carried out in the original paper (Arjona et al., 2014) (Figure 3A). We have added the following text:

“Several point mutations in CNNM2 have been identified in human patients with hypomagnesemia, which is associated with brain malformation and intellectual disability (Arjona et al., 2014). […] Staining for an associated C-terminal HA-tag revealed clear expression of all UAS-CNNM2::HA variants in αβ neurons when driven with c739-GAL4 (Figure 5—figure supplement 1).”

8) The impact of the T626NRR mutation on protein function is unclear, raising the concern that it could have a broader impact on the protein than simply altering nucleotide binding domain function. Is the mutant protein expressed and localized properly? Does the mutant protein have altered cAMP binding? Does it affect magnesium efflux activity? Barring massive experiments, a repeat of their MagIC experiment from Figure 8 using that specific construct would be satisfactory. If the authors do not know what the biochemical impact of this lesion is and are unable to carry out this experiment at present, they should insert appropriate caveats when interpreting the phenotype of this mutant.

These are important issues that we have now addressed in the manuscript. We have checked the expression of the T626NRR mutant protein via western blot and have added the data as panel E of Figure 6. The proteins all appear to be expressed at comparable levels in the KCs. The figure legend is updated accordingly. We also attempted MagIC experiments using the T626NRR mutant but have so far failed to generate flies carrying all the transgenes in the T626NRR mutant background. Text describing these data and stating caveats is now added:

“Although we confirmed using western blotting that a full-length protein is expressed in uex^T622NRR flies (Figure 6E), our antibody did not permit us to verify that the UEX^T626NRR protein localizes appropriately in the brain. Further work is therefore required to characterize the cellular localization, cAMP binding and Mg²⁺ transport function of the protein encoded by this serendipitous uex^T626NRR allele.”

Also, a subsection “Anti-UEX antibody and Western blot” has been added in the Materials and methods section of the manuscript.

9) Why were the HEK cell experiments done with a human CNNM4 while the fly experiments were done with human CNNM2? They're homologous, but why not use the same protein in each experiment to strengthen the parallels.

We emphasized conservation between UEX in the fly and mammalian CNNM2/4. In the HEK293 cell experiments, the human CNNM4 construct was only used as a positive control, and there was no intention to compare the extent of Mg²⁺ extrusion activity of CNNM4 and UEX in detail because we are currently unsure if their expression is comparable in HEK cells. Moreover, similar extrusion activity of CNNM2 and CNNM4 was previously established in Figure 1 of Hirata et al., 2014. We have added the following sentence:

“Mg²⁺ extrusion driven by UEX was noticeably less efficient than in cells expressing Human CNNM4 (Figure 5—figure supplement 2B), which is known to have similar efficiency to CNNM2 (Hirata et al., 2014). […] Nevertheless, demonstration of cross-species complementation and Mg²⁺ efflux activity defines UEX as a functional homolog of mammalian CNNM2/4.”

Author details

1. Yanying Wu

   Centre for Neural Circuits and Behaviour, The University of Oxford, Tinsley Building, Oxford, United Kingdom

   Contribution

   Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

2. Yosuke Funato

   Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Suita, Japan

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Methodology

   Competing interests

   No competing interests declared

3. Eleonora Meschi

   Centre for Neural Circuits and Behaviour, The University of Oxford, Tinsley Building, Oxford, United Kingdom

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2401-7969

4. Kristijan D Jovanoski

   Centre for Neural Circuits and Behaviour, The University of Oxford, Tinsley Building, Oxford, United Kingdom

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

5. Hiroaki Miki

   Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Suita, Japan

   Contribution

   Resources, Supervision, Funding acquisition, Methodology

   Competing interests

   No competing interests declared

6. Scott Waddell

   Centre for Neural Circuits and Behaviour, The University of Oxford, Tinsley Building, Oxford, United Kingdom

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   scott.waddell@cncb.ox.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4503-6229

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