Part 2:Magnesium Efflux From Drosophila Kenyon Cells Is Critical For Normal And Diet-enhanced Long-term Memory
Mar 17, 2022
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A role for uex in the mushroom bodies
To localize uex in the brain we first took advantage of VT23256-GAL4 transgenic flies, in which GAL4 is driven by an 853 bp sequence from the first intron of uex (Kvon et al., 2014). VT23256-driven UAS-EGFP revealed restricted expression in ab KCs with particularly strong label in ab core (abc) neurons (Figure 3A). We also used CRISPR to insert a C-terminal HA-epitope tag into the uex open reading frame (Figure 3—figure supplement 1A). These flies were viable as homozygotes indicating that the resulting UEX::HA fusion protein retains function. Immunostaining flies harboring this uex:: HA locus with an anti-HA antibody revealed prominent labeling of all the major KC classes in the MB, in addition to lower expression throughout the brain (Figure 3B). This uex expression profile is also supported by single-cell sequencing analyses (Figure 3—figure supplement 1B; Croset et al., 2018; Davie et al., 2018). Given the established role for ab KCs in olfactory LTM (Pascual and Pre´at, 2001; Yu et al., 2006; Krashes et al., 2007; Krashes and Waddell, 2008), we reasoned that a mnemonic role for UEX may involve expression in KCs.
We next used GAL4-directed expression of RNAi to test whether 24 hr memory performance required uex in the MB. Flies expressing uexRNAi (Perkins et al., 2015) in all ab KCs (c739-GAL4; Yang et al., 1995; Perisse et al., 2013) or only in abc KCs (NP7175-GAL4; Tanaka et al., 2008) showed normal immediate memory but significantly impaired 24 hr memory (Figure 3C). In contrast, uexRNAi expression in ab surface (abs, 0770-GAL4; Parisse et al., 2013) or a0 b0 KCs (c305a-GAL4; Krashes et al., 2007) did not significantly alter immediate or LTM performance. Normal 24 hr appetitive memory performance is therefore particularly sensitive to uex expression in abc neurons. To reduce the likelihood that the uexRNAi associated memory defect results from a developmental consequence, we also restricted UAS-uexRNAi expression to adulthood using GAL80ts-mediated temporal control (McGuire et al., 2003). At permissive 18˚C, GAL80ts binds to GAL4 and suppresses its transcriptional activator function. At restrictive 30˚C, GAL80ts can no longer bind to GAL4, which frees GAL4 to direct expression of the UAS-uexRNAi transgene. Flies were raised through development at 18˚C and moved to 30˚C after eclosion. Restricting UAS-uexRNAi expression to ab KCs in adult flies using c739-GAL4 with GAL80ts produced a similar 24 hr specific memory defect to that observed when UAS-uexRNAi was expressed without temporal control (Figure 3D–F). We assessed the efficacy of the UAS-uexRNAi knockdown using our tagged uex::HA locus. Brains from heterozygous uex::HA flies expressing uexRNAi in the ab and g KCs with MB247-GAL4 (Zars et al., 2000) were immunostained using anti-HA antibody. Comparing the intensity of immunolabeling in brains from uex::HA; MB247-GAL4/uexRNAi flies with that from uex::HA; MB247-GAL4/+ flies showed that uexRNAi expression significantly reduced anti-HA signal in the ab and g KCs (Figure 3G and H). This result demonstrates the efficiency of the uexRNAi transgene and the utility of the CRISPR/Cas9 edited uex::HA locus.
We next tested whether expression in specific KCs of an UAS-uex transgene could restore 24 hr memory capacity to uexMI01943 flies. Memory performance of uexMI01943 flies expressing UAS-uex in ab and g KCs (MB247-GAL4; Zars et al., 2000) or only the ab KCs (c739-GAL4) was significantly improved over that of uexMI01943 flies and was statistically indistinguishable from that of controls with an intact uex locus (Figure 4A). In contrast, UAS-uex expression in a0 b0 , abc, or abs KCs did not restore memory performance to uexMI01943 flies, and overexpressing uex in ab KCs of wild-type flies did not augment 24 hr memory (Figure 4A and B). Normal 24 hr memory performance could also be restored to uexMI01943 flies if UAS-uex expression was confined to c739-GAL4 neurons (all ab KCs) in adulthood using GAL80ts-mediated temporal control (Figure 4C and D). Together, these loss-of-function RNAi and restoration experiments establish that UEX plays an important role in adult
Figure 3. Knocking down uex expression in ab Kenyon cells (KCs) impairs LTM. (A) A uex promoter fragment-GAL4 directs GFP expression in abc KCs. Anti-GFP immunostained uex-GAL4 (VT23256); UAS-EGFP line. (B) Anti-HA immunostaining of brains harboring the CRISPR/Cas9-edited uex::HA locus
shows strong labeling of UEX in all the major subdivisions of the mushroom body (MB). Scale bars 20 mm. (C) RNAi knockdown of uex in all ab (c739- GAL4) or just abc (NP7175-GAL4) KCs specifically impaired 24 hr memory. abs (0770-GAL4) or a0 b0 (c305a-GAL4) KC expression had no effect (p<0.05, Figure 3 continued on next-Figure 3 continued
ANOVA, n = 6–10 for immediate and n = 8–14 for 24 hr memory). (D) Defective LTM was observed if uexRNAi expression was confined to ab KCs of adult flies using GAL80ts-mediated temporal control. (E) LTM performance was unaffected if the uexRNAi was kept suppressed throughout and (F) LTM performance was restored to normal levels if the expression of uexRNAi was re-suppressed for 3 days (p<0.05, ANOVA, n = 6 for immediate and n = 8 for 24 hr memory). (G) Immunostaining shows the effectiveness of uexRNAi. Fluorescence intensity in the ab and globes of uex::HA flies decreased significantly when UAS-uexRNAi was expressed with MB247-GAL4. Scale bars 20 mm. (H) Quantification of fluorescence intensity in G (p<0.05, t-test, n = 6–8). The online version of this article includes the following figure supplement(s) for figure 3:
Figure supplement 1. Construction scheme for the uex::HA line and tSNE plots of uex expression.
ab KCs. Finding that abc RNAi knockdown of uex produces a memory defect (Figure 3C) but UAS- uex expression in abc does not rescue the uexMI01943 mutant defect (Figure 4A) suggests that UEX function in abc KCs is essential for appetitive LTM, whereas both the abc and abs KCs need to have functional UEX to support LTM. In addition, the ability of UAS-uex to restore performance to uexMI0194 flies provides further support that uex is responsible for the memory impairment in uexMI01943 flies.
uex expression in the MB supports Mg2+-enhanced memory
We next investigated whether Mg2+ feeding (4 days with 80 mM MgCl2) could improve memory performance in flies with compromised uex function. Flies carrying the uexMI01943 allele (Figure 4F) or those expressing UAS-uexRNAi in the ab KCs with c739-GAL4 (Figure 4E) did not show enhanced memory when fed with 80 mM MgCl2, as compared to flies fed with 1 mM MgCl2. Moreover, the Mg2+-enhanced memory was recovered in uexMI01943 mutant flies when uex expression was restored to the ab KCs (Figure 4F). All control flies (c739-GAL4, UAS-uexRNAi, and UAS-uex) with unperturbed uex expression exhibited significantly enhanced memory when fed with 80 mM as compared to 1 mM MgCl2. Overexpressing UAS-uex in ab KCs with c739-GAL4 in flies with a wild-type genetic background neither enhanced regular 24 hr memory (Figure 4B), or that in flies fed for 4 days with 40 or 80 mM MgCl2 (Figure 4G). We also tested whether 4 days of 80 mM MgCl2 supplementation enhanced 24 hr memory performance following aversive spaced training. Again, the memory of wild- type, but not uexMI01943 mutant flies showed enhancement (Figure 4—figure supplement 1). Together these results indicate that optimal memory enhancement with Mg2+ feeding requires, and can be fully supported by, UEX function in ab KCs.
UEX is a functionally conserved magnesium transporter
Given the strong sequence conservation of UEX with mammalian CNNM2/4 we tested whether CNNM2 could functionally substitute for UEX and restore the LTM defect of uexMI01943 flies. Several point mutations in CNNM2 have been identified in human patients with hypomagnesemia, which is associated with a brain malformation and intellectual disability (Arjona et al., 2014). Introduction of the equivalent mutations into mouse CNNM2 (CNNM2E357K, CNNM2T568I, CNNM2S269W, and CNNM2E122K) showed that these patient-derived lesions impair magnesium transport (Arjona et al., 2014). We constructed flies carrying wild-type and this mutant variant UAS-CNNM2 transgenes (Figure 5A). Staining for an associated C-terminal HA-tag revealed clear expression of all UAS- CNNM2::HA variants in ab neurons when driven with c739-GAL4 (Figure 5—figure supplement 1). However, only expression of wild-type CNNM2, and not point-mutant forms, in ab KCs of uexMI01943
mutant flies restored 24 hr memory performance (Figure 5B).
We also tested whether UEX can mediate Mg2+ extrusion. UEX expressed in HEK293 cells localized to the plasma membrane and cells loaded with Mg2+ and the Mg2+ indicator Magnesium Green showed rapid Mg2+ efflux (Figure 5—figure supplement 2 and Video 1), as compared to cells transfected with empty vector. Mg2+ extrusion driven by UEX was noticeably less efficient than in cells expressing Human CNNM4 (Figure 5—figure supplement 2), which is known to have similar efficiency to CNNM2 (Hirata et al., 2014). However, we do not know if UEX and CNNM4 expression is equivalent. Nevertheless, demonstration of cross-species complementation and Mg2+ efflux activity defines UEX as a functional homolog of mammalian CNNM2/4.
Figure 4. Rescue of the LTM defect in uexMI01943 flies. Restoring expression of UAS-uex in ab and g (MB247-GAL4) or ab Kenyon cells (KCs) rescued 24 hr memory performance of uexMI01943 flies, whereas expression in abc, abs or a0 b0 KCs did not (p<0.05, ANOVA and t-test, n = 8–12). (B) Overexpression of UAS-uex in ab KCs did not enhance 24 hr memory performance in wild-type flies (ANOVA, n = 8–12). (C) Defective LTM was rescued if UAS-uex expression was confined to ab KCs of adult flies using GAL80ts mediated temporal control (p<0.05, ANOVA, n = 6 for immediate and n = 8 Figure 4 continued on next
for 24 hr memory) but (D) remained defective if UAS-uex expression was not released. (E) Memory enhancement with dietary Mg2+ is supported by UEX in ab KCs. The memory of flies expressing UAS-uexRNAi in the ab KCs cannot be enhanced with Mg2+ feeding (t-test, n = 8). (F) The memory of uexMI01943 mutant flies cannot be enhanced with Mg2+ feeding, but enhancement was restored by expressing UAS-uex in ab KCs (p<0.05, t-test, n = 8–12). (G) The memory of wild-type flies was not sensitized to Mg2+ enhancement by overexpressing UAS-uex in ab KCs. Memory was enhanced if the flies were fed with 80 mM MgCl2, but not with suboptimal 40 mM MgCl2 (p<0.05, ANOVA, n = 8).
The online version of this article includes the following figure supplement(s) for figure 4:
Figure supplement 1. Mg2+ feeding enhanced LTM after aversive spaced training in wild-type but not uexMI01943 mutant flies.
An intact CNBH domain is required for memory
Given the established role for cAMP signaling in memory-relevant plasticity in invertebrates and mammals (Kandel, 2012), we tested the importance of the CNBH domain in UEX. We constructed flies carrying a point-mutated CNBH UAS-uexR622K transgene (Figure 6A). The equivalent R622K amino acid substitution abolishes cAMP binding in the regulatory subunit of cAMP-dependent protein kinase, PKA (Bubis et al., 1988). Expressing UAS-uexR622K in ab neurons with c739-GAL4 did not restore 24 hr memory performance, or alter the immediate memory performance, of uexMI01943 mutant flies (Figure 6B).
We also used CRISPR to attempt to introduce the R622K mutation into the CNBH of the native uex locus (Bassett et al., 2013; Gratz et al., 2013; Yu et al., 2013). Unexpectedly, this approach did not introduce the R622K substitution but instead replaced T626 in the CNBH with NRR. Fortuitously, flies homozygous for this uexT626NRR allele were viable as adults, unlike those homozygous for uexD, suggesting that the uexT626NRR encoded UEX retains function. However, flies homozygous for uexT626NRR or heterozygous uexT626NRR/ uexMI01943 flies exhibited a strong 24 hr memory defect (Figure 6C). Immediate memory was also impaired in homozygous uexT626NRR flies, unlike flies carry- ing all other combinations of uex alleles. In addition, the memory of uexT626NRR flies could not be enhanced with Mg2+ feeding (Figure 6D). The uexT626NRR mutation, therefore, uncouples the essential
Figure 5. uex encodes an evolutionarily conserved Mg2+ transporter. (A) Model of CNNM2 protein structure showing clinically relevant point mutations.
Adapted and modified from Arjona et al., 2014. (B) Overexpression of wild-type, but not mutant, CNNM2 in ab Kenyon cells rescues the memory defect of uexMI01943 mutant flies (p<0.05, ANOVA, n = 6–8 for immediate and n = 8–12 for 24 hr memory).
The online version of this article includes the following figure supplement(s) for figure 5:
Figure supplement 1. Transgenic expression of mutant variants of CNNM2.
Figure supplement 2. UEX-dependent Mg2+ efflux in HEK293 cells.
role for uex from a function in memory and sug- gests that cyclic nucleotide regulated activity is critical for UEX to support normal and Mg2+- enhanced memory. Although we confirmed using western blotting that a full-length protein is expressed in uexT622NRR flies (Figure 6E), our antibody did not permit us to verify that the UEXT626NRR protein localizes appropriately in the brain. Further work is therefore required to char- acterize the cellular localization, cAMP binding, and Mg2+ transport function of the protein encoded by this serendipitous uexT626NRR allele.
Chronic cAMP manipulation alters UEX localization in KCs
We tested whether cAMP could acutely alter UEX activity by applying forskolin to UEX- expressing HEK293 cells. However, no obvious change in the UEX-dependent Mg2+ efflux dynamic was observed (data not shown). We therefore tested whether KC expression of UEX:: HA was altered in flies with chronic alterations of
cAMP metabolism, by introducing learning-rele- vant mutations in the rutabaga-encoded Ca2+- stimulated adenylate cyclase, or the dunce-encoded cAMP-specific phosphodiesterase. Anti-HA immunostaining of brains from rut2080; uex::HA and dnc1; uex::HA flies revealed a striking change in UEX localization (Figure 7A and B and Videos 2–4). Whereas UEX::HA is usually detected in the lobes of all KCs at a roughly equivalent level in wild-type flies, labeling was lower in the MB g lobe and more pronounced in the abc KCs in rut2080 and dnc1 mutant backgrounds (Figure 7C), although the overall MB expression of UEX::HA is similar between wild-type and mutant flies (Figure 7D). In addition, western blot analyses of protein extracted from heads of these flies did not reveal a significant difference in overall UEX::HA expression levels (data not shown). These data are therefore consistent with cAMP regulating UEX function and perhaps its cellular localization in KCs.
UEX is required to maintain a fluctuating [Mg2+]i in ab KCs
Although MagFRET can report [Mg2+] it does not respond quickly enough to record stimulus-evoked signals. We therefore constructed flies harboring UAS-transgenes for two newer genetically encoded Mg2+ sensors, MagIC (non-FRET based; Koldenkova et al., 2015) and MARIO (FRET based; Maeshima et al., 2018). We were unable to detect UAS-MARIO expression in the fly brain and therefore could only use UAS-MagIC. MagIC was reported to respond most strongly to Mg2+ but also to a lesser extent to Ca2+ (Koldenkova et al., 2015). We therefore first verified the specificity of MagIC responses in a cell-permeabilized ex vivo fly brain preparation. Brains were removed from flies expressing UAS-MagIC in ab KCs with c739-GAL4 (Figure 8A), incubated in a dish with saline (Barnstedt et al., 2016) and changes in fluorescence were monitored before and after bath applica- tion of chemicals. Whereas application of MgCl2 evoked a dose-dependent increase in the MagIC response, chelation of Mg2+ with EDTA produced a dose-dependent decrease (Figure 8B and Vid- eos 5 and 6). In comparison, CaCl2 only registered a slight increase at the highest concentrations whereas the more Ca2+-selective chelator EGTA had little effect (Figure 8B). These results demon- strate that UAS-MagIC can monitor [Mg2+]i in the ab KCs in the fly brain.
Increasing intracellular cAMP has been shown to elicit Mg2+ flux from mammalian cells (Romani and Scarpa, 2000; Vormann and Gu¨nther, 1987; Jakob et al., 1989; Romani and Scarpa, 1990b; Romani and Scarpa, 1990a; Vormann and Gu¨nther, 1987; Gu¨nther et al., 1990; Howarth et al., 1994). Since our experiments also indicated that cAMP might regulate UEX, we next tested whether stimulating cAMP synthesis with forskolin (FSK) might alter MagIC signals in ab
Figure 6. The cyclic nucleotide-binding homology (CNBH) domain of UEX is required for memory. (A) Schematic showing sequence detail of the CNBH domain in UEX, and the amino acid changes made in uexR622K and uexT626NRR. (B) Expressing a UAS-uexR622K transgene in ab Kenyon cells did not rescue the LTM defect of uexMI01943 mutant flies (p<0.05, ANOVA, n = 8). Immediate memory was also unaffected. (C) Flies homozygous for uexT626NRR have defective short- and long-term memory, while trans-heterozygous uexT626NRR/uexMI01943 flies only exhibit impaired LTM (*p<0.05, ANOVA, n = 8). Figure 6 continued on next page
Figure 6 continued
(D) Dietary Mg2+ did not enhance memory of homozygous uexT626NRR/ uexT626NRR flies (p<0.05, t-test, n = 8). (E) Western blot analysis of UEX protein expression in fly head extracts. Genotype from left to right: wild-type, uexT626NRR/uexT626NRR, uexT626NRR/+, uexT626NRR/uexMI01943, uexMI01943/+. The blot was first probed with anti-UEX antibody (upper panel), and then stripped and re-probed with anti-Tubulin antibody (lower panel) as a loading control.
KCs. For these experiments we again used an ex vivo brain preparation but this time the cells were not permeabilized. 30 mM FSK has been shown to evoke a peak increase in cAMP in KCs that approximates that observed following appetitive conditioning (Louis et al., 2018). Applying 30 mM FSK to c739-GAL4; UAS-MagIC brains evoked a consistent dynamic in MagIC fluorescence. After a sharp initial rise, responses slowly decayed back toward baseline before again rising slowly to a point at which the signal started to fluctuate. (Figure 8C and D and Video 7). The key signatures of this response were only recorded in the Mg2+-sensitive Venus signal (Figure 8D). In contrast mCherry fluorescence did not fluctuate but steadily decreased across the time course of the recording (likely a result of photo-bleaching), demonstrating that the fluctuation in the Venus signal is not a move- ment artifact (Figure 8E). Importantly, FSK induced MagIC responses were greater than those fol- lowing application of saline (Figure 8—figure supplement 1A). However, a fluctuating response also developed after saline applications (Figure 8—figure supplement 1B) suggesting that the rhythmic MagIC signal may be a general response to an increase in [Mg2+]i that follows cellular perturbation.
The Drosophila MB has previously been reported to exhibit a slow (0.004 Hz) Ca2+ oscillation in ex vivo brains whereas a much faster 20 Hz oscillation is evoked by odors in the locust MB (Laurent and Naraghi, 1994; Rosay et al., 2001). Although our initial characterization of MagIC in the fly brain indicated a preferential response to Mg2+ (Figure 8B), we nevertheless explicitly tested whether FSK induced fluctuation of the [Ca2+]i of ab KCs, using expression of UAS-GCaMP6f (Chen et al., 2013). FSK induced a delayed increase in the GCaMP response but no clear oscillatory activity was observed (Figure 8—figure supplement 1C–E).
Lastly, we tested whether the observed MagIC responses were sensitive to the status of the uex gene. We generated uexMI01943 flies that also harbored c379-GAL4 and UAS-MagIC and compared their FSK- and saline-induced MagIC responses to those of flies with a wild-type uex locus. The uexMI01943 mutant flies showed an increased FSK response to that of wild-type flies, whereas saline- evoked responses were indistinguishable (Figure 8F and G). Responses evoked by the inactive FSK analogue, ddFSK, were also insensitive to the status of uex (Figure 8—figure supplement 1F). Mutation of uex therefore selectively increases mean FSK-evoked MagIC responses.
We also noticed that MagIC traces from uex mutant flies did not exhibit a fluctuating signal (Figure 8H and Figure 8—figure supplement 1G). To quantify this difference we calculated the mean power spectral density (PSD) of traces from uexMI01943 and wild-type flies treated with FSK or saline. In both conditions the mean PSD was significantly left-shifted toward lower frequencies in the uexMI01943 mutants compared to the wild-type controls (Figure 8I). Wild-type fly brains had signifi- cantly more oscillatory activity centered around 0.015 Hz than those from uexMI01943 mutants. These data therefore suggest that UEX is required for slow rhythmic maintenance of KC [Mg2+]i. Impor- tantly, finding that MagIC signals are elevated and altered in uex mutants confirms that the observed MagIC responses are Mg2+-dependent. Moreover, they suggest that the KC expressed UEX limits Mg2+ accumulation, consistent with a role in extrusion.
Discussion
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 magne- sium chloride, magnesium sulfate, or MgT (data not shown).
Figure 7. Kenyon cell (KC) uex expression is altered in rutabaga and dunce mutant flies. (A) Anti-HA stained brains reveal UEX::HA protein localization is altered in rut2080; uex::HA and dnc1; uex::HA flies, becoming more prominent in abc KCs (arrows). Scale bars 20 mm. (B) Enlarged images of the mushroom bodies (MBs) highlighting abc KC expression in rut2080 and dnc1 mutant flies, as compared with wild-type uex::HA flies. Scale bars 20 mm. (C) Quantification of fluorescence intensity. Left, micrograph with a measurement line through the a lobe tip and rectangular ROIs for the g lobe and a control area. Middle, relative fluorescence intensity profiles across the lobe tip show significantly higher signal in rut2080 and dnc1 mutant flies in the center region occupied by the ab core KCs (*p<0.05, ANOVA, n = 6–10). Right, the relative intensity in the g lobe was significantly lower in rut2080 and dnc1 mutant flies, as compared to wild-type controls (*p<0.05, ANOVA, n = 6–10). Scale bars 10 mm. (D) Left, micrograph showing circular ROIs. Right, Figure 7 continued on quantification. Total intensity over all six ROIs on the MBs was not significantly different between the rut2080, dnc1, and wild-type brains (p>0.13; ANOVA, n = 6–10).
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 pre- viously 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 NMDARN631Q 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; Parisse et al., 2016; Aso et al., 2014; Owald and Wad- dell, 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 ab KCs by approx-
mately 50 mM.
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; Gime´nez-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 ab KCs in the fly brain. Therefore, if UEX is not itself an 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 indi- vidual 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; Gime´nez-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 uexD 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 uexRNAiwith 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). Per- haps 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 ab KCs of adult flies, or even just the abc subset reproduced the LTM defect. The LTM impairment was evident if uexRNAi expression in ab neurons
Figure 8. UEX limits a rise in [Mg2+]i and supports a slow oscillation in ab Kenyon cells (KCs). (A) Explant fly brain expressing UAS-MagIC driven by c739-GAL4. Upper panel, wide-field phase contrast view; middle panels, fluorescence views of Venus and mCherry channels; lower panel, a confocal section at the level of the KC somata showing Venus and mCherry channels. Scale bars 20 mm. (B) MagIC selectively responds to changes in [Mg2+]i in KCs. Traces of MagIC ratio following bath application of 10, 20, or 40 mM MgCl2 or CaCl2; 5, 10, or 20 mM EDTA or EGTA. (C) Representative trace of Figure 8 continued on next -Figure 8 continued
MagIC ratio following the application of FSK shows an initial wave followed by a gradual rise and the development of a slow oscillation. (D) The primary responses result from changes in the Mg2+-sensitive Venus signal. (E) The mCherry signal exhibits a steady decay. (F) FSK-evoked MagIC responses are greater in uex mutant flies. Averaged MagIC responses show that FSK induced a significantly greater increase in uexMI01943 mutant than in wild-type flies. (G). Averaged saline-evoked MagIC responses were not significantly altered in uex mutant flies. (H) Individual Venus (green) and mCherry (red) channel traces show that the slow oscillation is only evident in the Venus channel of wild-type, but not uex mutant, flies. (I) Power spectral density (PSD) analysis of the time series from 200 to 900 s of all data shows that traces from wild-type flies have significantly more oscillatory activity, centered around 0.015 Hz, than those from uex mutant flies.
The online version of this article includes the following figure supplement(s) for figure 8:
Figure supplement 1. Individual traces for MagIC and GCaMP imaging.
