InteractiveFly: GeneBrief

unextended : Biological Overview | References

Dietary magnesium (Mg(2+)) supplementation can enhance memory in young and aged rats. Memory-enhancing capacity was largely ascribed to increases in hippocampal synaptic density and elevated expression of the NR2B subunit of the NMDA-type glutamate receptor. This study shows that Mg(2+) feeding also enhances long-term memory in Drosophila. Normal and Mg(2+) enhanced fly memory appears independent of NMDA receptors in the mushroom body and instead requires expression of a conserved CNNM-type Mg(2+)-efflux transporter encoded by the unextended (uex) gene. UEX contains a putative cyclic nucleotide-binding homology domain and its mutation separates a vital role for uex from a function in memory. Moreover, UEX localization in mushroom body Kenyon Cells is altered in memory defective flies harboring mutations in cAMP-related genes. Functional imaging suggests that UEX-dependent efflux is required for slow rhythmic maintenance of Kenyon Cell Mg(2+). It is proposed that regulated neuronal Mg(2+) efflux is critical for normal and Mg(2+) enhanced memory (Wu, 2020).

Magnesium (Mg2+) plays a critical role in cellular metabolism and is considered to be an essential co-factor for more than 350 enzymes. 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, Parkinson's disease, Alzheimer's disease, anxiety, depression, and intellectual disability (Wu, 2020).

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. In addition, elevated Mg2+ reduced cognitive deficits in a mouse model of Alzheimer's disease and enhanced the extinction of fear memories. These apparently beneficial effects have led to the proposal that dietary Mg2+ may have therapeutic value for patients with a variety of memory-related (Wu, 2020).

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 (see Drosophila NMDA receptors) and thereby inhibits the passage of other ions. 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. 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. The enhancing effects of increased [Mg2+]e were also observed in vivo in the brain of rats fed with Mg2+-L-threonate. Hippocampal neuronal circuits undergo homeostatic plasticity to accommodate the increased [Mg2+]e by upregulating expression of NR2B subunit containing NMDARs. 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 (Wu, 2020).

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). In addition, olfactory information is conveyed to KCs by cholinergic transmission from olfactory projection neurons. 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. The fly therefore provides a potential model to investigate other mechanisms through which dietary Mg2+ might enhance memory (Wu, 2020).

The reinforcing effects of dopamine depend on the Dop1R D1-type dopamine receptor, which is positively coupled with cAMP production. Moreover, early studies in Drosophila identified the dunce and rutabaga encoded cAMP phosphodiesterase and type I Ca2+-stimulated adenylate cyclase, respectively, to be essential for olfactory memory. 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. However, it is unclear whether Mg2+ extrusion plays any role in memory processing (Wu, 2020).

This study demonstrates that Drosophila long-term memory (LTM) can be enhanced with dietary Mg2+ supplementation. The unextended (uex) 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 (Wu, 2020).

This study 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. 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, a similar enhancement of memory performance was observed when flies were fed with magnesium chloride, magnesium sulfate, or MgT (Wu, 2020).

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 suggesting that this switch in subunit composition represents a homeostatic plasticity mechanism 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, 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, it was noted that NMDAR currents were insufficient to fully explain the observed changes (Wu, 2020).

NMDAR subunit loss-of-function studies in the Drosophila KCs did not impair regular or Mg2+-enhanced memory. Furthermore, no obvious change was detected 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. 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. 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. 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, MagFRET measurements indicate that Mg2+ feeding also increases the [Mg2+]i of αβ KCs by approximately 50 μM (Wu, 2020).

This study identified a role for uex, the single fly ortholog of the evolutionarily conserved family of CNNM-type Mg2+ efflux transporters (Ishii, 2016). There are four distinct CNNM genes in mice and humans, five in C. elegans, and two in zebrafish (Ishii, 2016; Arjona, 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. 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. This study 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 (Wu, 2020).

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. 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. It will therefore be of interest to test the role of the UEX CBS domains and whether fly PRL-1 regulates UEX activity (Wu, 2020).

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 suggesting that many uex mutations will be pleiotropic. The uex^Δ 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. 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^RNAi with elav-GAL4 also results in larval lethality, suggesting UEX has an additional role in early development of the nervous system, like CNNM2 in humans and zebrafish (Arjona, 2014; Accogli, 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 (Wu, 2020).

Genetically engineering the uex locus to add a C-terminal HA tag to the UEX protein allowed localization of 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 αβc 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 αβs or α'β' neurons did not impair LTM. Activity of α'β' neurons is required after training to consolidate appetitive LTM, whereas αβc and αβs KC output, together and separately, is required for its expression. Therefore, observing normal LTM performance in flies with uex loss-of-function in αβs and α'β' neurons argues against a general deficiency of αβ neuronal function when manipulating uex (Wu, 2020).

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 Mg2+-enhanced memory in the fly (Wu, 2020).

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, it can only be speculated as to why this is the case. It is assumed 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. 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 (Wu, 2020).

A number of mammalian cell-types extrude Mg2+ in a cAMP-dependent manner, a few minutes after being exposed to β-adrenergic stimulation. The presence of a CNBH domain suggests that UEX and CNNMs could be directly regulated by cAMP. The importance of the CNBH was tested 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. CRISPR was used to mutate the CNBH in the native uex locus. Although deleting the CNBH from CNNM4 abolished Mg2+ efflux activity (Chen, 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, 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, did not restore the uex LTM defect (Wu, 2020).

Although it has been questioned whether the CNNM2/3 CNBH domains bind cyclic nucleotides, this study 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 and dnc¹ phosphodiesterase learning defective mutant flies. Whereas UEX::HA label was evenly distributed in γ, αβc, and αβs KCs in wild-type flies, UEX::HA label was diminished in the γ and αβs KCs and was stronger in αβc 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 (Wu, 2020).

The 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. 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. The physiological function of this [Mg2+]i fluctuation is not yet understood 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. 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. It is therefore possible that slow Mg2+ oscillations could unite roles for cAMP, UEX, energy flux, and mTOR-dependent translation underlying LTM-relevant synaptic plasticity (Wu, 2020).

A Novel Neuroprotective Role of Phosphatase of Regenerating Liver-1 against CO2 Stimulation in Drosophila

Neuroprotection is essential for the maintenance of normal physiological functions in the nervous system. This is especially true under stress conditions. This study demonstrates a novel protective function of PRL-1 against CO2 stimulation in Drosophila. In the absence of PRL-1, flies exhibit a permanent held-up wing phenotype upon CO2 exposure. Knockdown of the CO2 olfactory receptor, Gr21a, suppresses the phenotype. Genetic data indicate that the wing phenotype is due to a neural dysfunction. PRL-1 physically interacts with Uex and controls Uex expression levels. Knockdown of Uex alone leads to a similar wing held-up phenotype to that of PRL-1 mutants. Uex acts downstream of PRL-1. Elevated Uex levels in PRL-1 mutants prevent the CO2-induced phenotype. PRL-1 and Uex are required for a wide range of neurons to maintain neuroprotective functions. Expression of human homologs of PRL-1 could rescue the phenotype in Drosophila, suggesting a similar function in humans (Guo, 2019).

This study has demonstrated that in Drosophila adult flies, PRL-1 functions in the nervous system and prevents CO2-induced neural defects manifested by a held-up wing phenotype. Genetic rescue data strongly indicate that it is defects in the nervous system that cause the CO2-induced wing phenotype in PRL-1 mutant flies. No obvious defects in muscles were observed, and ectopic expression of PRL-1 in muscles alone could not rescue the phenotype. The CO2-induced wing phenotype was triggered initially by the signals from the CO2 sensory neurons. Specific knockdown of CO2 receptor protein Gr21a in these neurons fully prevented the wing phenotype in the PRL-1 mutants (Guo, 2019).

The holding-up of wings in the fly is a behavioral output signal usually indicating avoidance or acceptance of a stimulus. Olfactory CO2 detection via the receptors Gr21a and Gr63a in the CO2-responsive neurons mediates avoidance, whereas E409 neurons, a population of taste neurons, have been identified to mediate taste acceptance in flies that are attracted to CO2 in solution. This indicates that compartmentalization of CO2 detection may allow Drosophila to distinguish local versus global CO2 levels and finely regulate behavior. In this study, the PRL-1 mutant flies retained normal responses to anesthesia including N2, volatile ether, and CO2. However, without PRL-1 protection, the processing of olfactory CO2 stimulation was rendered defective, resulting in a permanent holding up of wings. Many other gene mutations such as Apterous and Beadex could produce held-up curled wings. Parkin and pink mutants also exhibit held-up or drooped wing phenotype due to muscle defects. Such a hold up differs in nature to the PRL-1 case. This study reports that the occurrence of a held-up wing phenotype is caused by gene disruption, which might regulate neuronal homeostasis, and this demonstrates that olfactory CO2 stimulation is associated with the risk of neurological dysfunction for which PRL-1 provides defense (Guo, 2019).

Neural expression of shi^ts1 in wt background provides a valuable clue to understand the rationale behind the held-up wing phenotype. Within the recovery time (about 10 min) when flies were shifted back from the non-permissive temperature (29°C) to the permissive temperature (25°C), they exhibited a held-up wing phenotype, which was reminiscent of that observed in PRL-1 mutant flies induced by CO2 exposure, except that in this case it was transient rather than permanent. As the nervous system is only partially functional during the period of recovery, it is concluded that CO2-induced held-up wing phenotype in PRL-1 mutant flies is most likely due to neural dysfunction. These data showed that expression of shi^ts1 in motor neurons (D42-GAL4) also induced wing hold-up phenotype, although with a lower penetrance. This could simply be due to the lower-level expression of shi^ts1 in motor neurons (Guo, 2019).

There are three members of the mammalian PRL family. Drosophila PRL-1 shares high similarities (74%–76%) to all three mPRLs. It has been reported that PRL1/PRL2 double knockout mice were embryonic lethal. However, PRL1-/-/PRL2+/- and PRL1+/-/PRL2-/- mice are viable, suggesting that there is a functional redundancy between PRL1 and PRL2. Mice deficient for PRL3 were grossly normal. This study reveals that the PRL-1 mutant flies are viable and fertile, even when they occurred with held-up wings, which negated flight for their entire lifespan. Using molecular mapping, this study found that PRL-1 was enriched in the V-glomeruli of the AL and the MB of the Drosophila brain. This study demonstrates that PRL-1 functions to protect against olfactory CO2 stimulation. This study suggests that Drosophila PRL-1 might not be critically required for survival, but essential for the maintenance of the neural homeostasis under stress conditions (Guo, 2019).

In mammals, PRL-2 regulates intracellular magnesium levels by forming a functional heterodimer with the magnesium transporter CNNM3. However, a substrate-trapping assay revealed that the mutation of catalytic cysteine to serine, or the mutation of aspartic acid to alanine in the WPD motif of PRL-2, did not lead to increased complex formation but to a strong reduction in the binding between the two proteins. This suggests that a catalytically active form of PRL-2 is still crucial for its association with CNNM3. A similar result was obtained by using substrate trapping mutants in analyzing the binding of Drosophila PRL-1 to Uex and this study confirmed that Uex is not a typical phosphorylated substrate for PRL-1. The physiological substrate of PRl-1 is still unknown. It is possible that Drosophila PRL1 acts both as a trigger of Uex for a particular neuronal pathway and as a lipid phosphatase to maintain an active conformation for additional functions, for example, to control magnesium homoeostasis through the PRL-1/Uex complex (Guo, 2019).

The CBS pair domain of the magnesium transporter MgtE acts as a magnesium sensor and regulates the gating of the activity of the magnesium-transporting pore. To confirm that Uex protein does indeed bind PRL-1 through its CBS domain, a guide RNA targeted the CBS domain using CRISPR/Cas9 method. Many mutants were obtained, but most of them were lethal. Only one of them was homozygous viable, named uex7-7-1, which caused two amino acids to be turned to one amino acid in the CBS domain. The disrupted Uex protein extracted from this single mutant line exhibited decreased binding to PRL-1, as revealed by a GST pull-down assay (Guo, 2019).

In this study, loss of PRL-1 clearly decreased the expression of Uex. Direct knockdown of Uex resulted in the same wing phenotype as observed in the PRL-1 mutants, whereas abnormal wing posture in PRL-1 mutants could be restored by rescuing Uex expression, particularly in the nervous system. However, it was found that the loss of Uex causes fly lethality. In the mouse model, knockout of PRL-1 or PRL-2 only affects the related CNNMs protein. In this case, because the CNNM family has four members, the partial degradation of only one CNNM member is not enough to cause lethality. However, double mutants of PRL-1 and PRL-2 are clearly enough to decrease CNNMs' protein expression, which then causes the lethality of the mouse. Mg2+ acts as a physiological Ca2+ antagonist for blocking the excitatory N-methyl-D-aspartate receptors in the CNS and has therefore been suggested as a possible means of resolving muscle rigidity and spasms in cases of tetanus. In humans, mutations in CNNM2 cause seizures and mental retardation in patients with hypomagnesemia. CNNM4 can regulate Ca2+ influx during sperm capacitation. Although it was not possible to measure the Mg2+ homeostasis status in the PRL-1 mutants and uex-IR flies, enhanced Ca2+ activities were induced in the PRL-1 mutants. It would be possible that, if the cations, either magnesium or calcium, were added to the flies, this would affect the CNS homeostasis in Drosophila (Guo, 2019).

A complete rescue in the Drosophila PRL-1 wing phenotype was achieved by using either hPRL-1 or hPRL-2 transgenic flies. This may imply that human PRL phosphatases are poised to function in a way similar to that shown for neuroprotection in Drosophila. Human PRL-3 has been demonstrated to dephosphorylate lipids and to affect phosphatidylinositol 3-kinase (PI3K) signaling . Drosophila PRL-1 is also thought to affect phosphoinositide-dependent PI3K-PTEN signaling loop, leading to the spatially restricted synapse formation (Urwyler, 2019). For an unknown reason, a technical difficulty was found in producing hPRL-3 transgenic flies for the rescue experiment. Whether PRL1 in Drosophila acts as a lipid PTP (protein tyrosine phosphatase) in CO2 neural circuits remains to be illustrated (Guo, 2019).

In conclusion, this study has identified a novel neural protective function of PRL-1/Uex. In the absence of PRL-1, Uex expression levels are down-regulated. Upon CO2 exposure, the receptors in the CO2 sensory neuron send signals to the nervous system, triggering behavioral responses. The AL region of the brain in PRL-1 mutants exhibits hypersensitive Ca2+ responses to CO2 exposure. This hypersensitivity combined with low levels of Uex leads to neural dysfunction, resulting in the held-up wing phenotype. Although primarily recognized for PRL's oncogenic properties in mammals, this study highlights its neuroprotective role in the nervous system, particularly in relation to the CO2 sensory motor pathway in Drosophila. This study implies that PRLs may retain a similar neuroprotective function in humans. The phenomena of neurological dysfunction induced by CO2 insult in PRL-1 mutants resembles the post-traumatic stress disorder in humans, in which transient severe unfavorable stimulating factors cause ongoing neurological dysfunction. Further investigations are needed to confirm the correlation (Guo, 2019).

Although this study has revealed a Prl-1-Uex complex-based neuroprotective mechanism in which Prl-1 protects against nervous system insult related to olfactory CO2 stimulation, any human neuroprotective mechanisms related to the issue of CO2 toxicity, particularly those relating to olfactory pathways, have yet to be elucidated. It is true that in human brain disorders such as Parkinson and Alzheimer diseases, there is profound olfactory disorder in odor threshold detection, odor memory, or odor identification often occurring before disease onset. These are often associated with aspects of limb dysfunction. However, the reasons and mechanisms of such still remain unknown. The potential role of hPRL-1 in this process requires further study (Guo, 2019).

Essential loci in centromeric heterochromatin of Drosophila melanogaster. I: the right arm of chromosome 2

With the most recent releases of the Drosophila melanogaster genome sequences, much of the previously absent heterochromatic sequences have now been annotated. This study undertook an extensive genetic analysis of existing lethal mutations, as well as molecular mapping and sequence analysis (using a candidate gene approach) to identify as many essential genes as possible in the centromeric heterochromatin on the right arm of the second chromosome (2Rh) of D. melanogaster. Available RNA interference lines were used to knock down the expression of genes in 2Rh as another approach to identifying essential genes. In total, the existence of eight novel essential loci were identified in 2Rh: CG17665, CG17683, CG17684, CG17883, CG40127, CG41265, CG42595, and Atf6. Two of these essential loci, CG41265 and CG42595, are synonymous with the previously characterized loci l(2)41Ab and unextended, respectively. The genetic and molecular analysis of the previously reported locus, l(2)41Ae, revealed that this is not a single locus, but rather it is a large region of 2Rh that extends from unextended (CG42595) to CG17665 and includes four of the novel loci uncovered in this study (Coulthard, 2010).

The cyclic nucleotide-binding homology domain of the integral membrane protein CNNM mediates dimerization and is required for Mg(2+) efflux activity

Proteins of the cyclin M family (CNNMs; also called ancient conserved domain proteins, or ACDPs) are represented by four integral membrane proteins that have been proposed to function as Mg(2+) transporters. CNNMs are associated with a number of genetic diseases affecting ion movement and cancer via their association with highly oncogenic phosphatases of regenerating liver (PRLs). Structurally, CNNMs contain an N-terminal extracellular domain, a transmembrane domain (DUF21), and a large cytosolic region containing a cystathionine-beta-synthase (CBS) domain and a putative cyclic nucleotide-binding homology (CNBH) domain. Although the CBS domain has been extensively characterized, little is known about the CNBH domain. This study determined the first crystal structures of the CNBH domains of CNNM2 and CNNM3 at 2.6 and 1.9 Å resolutions. Contrary to expectation, these domains did not bind cyclic nucleotides, but mediated dimerization both in crystals and in solution. Analytical ultracentrifugation experiments revealed an inverse correlation between the propensity of the CNBH domains to dimerize and the ability of CNNMs to mediate Mg(2+) efflux. CNBH domains from active family members were observed as both dimers and monomers, whereas the inactive member, CNNM3, was observed only as a dimer. Mutational analysis revealed that the CNBH domain was required for Mg(2+) efflux activity of CNNM4. This work provides a structural basis for understanding the function of CNNM proteins in Mg(2+) transport and associated diseases (Chen, 2018).

CNNM2 homozygous mutations cause severe refractory hypomagnesemia, epileptic encephalopathy and brain malformations

Magnesium (Mg(2+)) plays a crucial role in many biological processes especially in the brain, heart and skeletal muscle. Mg(2+) homeostasis is regulated by intestinal absorption and renal reabsorption, involving a combination of different epithelial transport pathways. Mutations in any of these transporters result in hypomagnesemia with variable clinical presentations. Among these, CNNM2 is found along the basolateral membrane of distal tubular segments where it is involved in Mg(2+) reabsorption. To date, heterozygous mutations in CNNM2 have been associated with a variable phenotype, ranging from isolated hypomagnesemia to intellectual disability and epilepsy. The only homozygous mutation reported so far, is responsible for hypomagnesemia associated with a severe neurological phenotype characterized by refractory epilepsy, microcephaly, severe global developmental delay and intellectual disability. This study reports the second homozygous CNNM2 mutation (c.1642G7gt;A,p.Val548Met) in a Moroccan patient, presenting with hypomagnesemia and severe epileptic encephalopathy. Thus, the phenotypic spectrum associated with CNNM2 mutations is reviewed and discussed (Accogli, 2019).

Mg2+ extrusion from intestinal epithelia by CNNM proteins is essential for gonadogenesis via AMPK-TORC1 signaling in Caenorhabditis elegans

Mg2+ serves as an essential cofactor for numerous enzymes and its levels are tightly regulated by various Mg2+ transporters. This study analyzed Caenorhabditis elegans strains carrying mutations in genes encoding cyclin M (CNNM) Mg2+ transporters. Inactivating mutants were isolated for each of the five Caenorhabditis elegans cnnm family genes, cnnm-1 through cnnm-5. cnnm-1; cnnm-3 double mutant worms showed various phenotypes, among which the sterile phenotype was rescued by supplementing the media with Mg2+. This sterility was caused by a gonadogenesis defect with severely attenuated proliferation of germ cells. Using this gonadogenesis defect as an indicator, genome-wide RNAi screening was performed, to search for genes associated with this phenotype. The results revealed that RNAi-mediated inactivation of several genes restores gonad elongation, including aak-2, which encodes the catalytic subunit of AMP-activated protein kinase (AMPK). Triple mutant worms were generated for cnnm-1; cnnm-3; aak-2, and it was confirmed that the aak-2 mutation also suppressed the defective gonadal elongation in cnnm-1; cnnm-3 mutant worms. AMPK is activated under low-energy conditions and plays a central role in regulating cellular metabolism to adapt to the energy status of cells. Thus, this study providesr genetic evidence linking Mg2+ homeostasis to energy metabolism via AMPK (Ishii, 2016).

Identification and mechanistic analysis of an inhibitor of the CorC Mg(2+) transporter

The CorC/CNNM family of Na(+)-dependent Mg(2+) transporters is ubiquitously conserved from bacteria to humans. CorC, the bacterial CorC/CNNM family of proteins, is involved in resistance to antibiotic exposure and in the survival of pathogenic microorganisms in their host environment. The CorC/CNNM family proteins possess a cytoplasmic region containing the regulatory ATP-binding site. CorC and CNNM have attracted interest as therapeutic targets, whereas inhibitors targeting the ATP-binding site have not been identified. This study performed a virtual screening of CorC by targeting its ATP-binding site, identified a compound named IGN95a with inhibitory effects on ATP binding and Mg(2+) export, and determined the cytoplasmic domain structure in complex with IGN95a. Furthermore, a chemical cross-linking experiment indicated that with ATP bound to the cytoplasmic domain, the conformational equilibrium of CorC was shifted more toward the inward-facing state of the transmembrane domain. In contrast, IGN95a did not induce such a shift (Huang, 2021b).

Structural basis for the Mg(2+) recognition and regulation of the CorC Mg(2+) transporter

The CNNM/CorC family proteins are Mg(2+) transporters that are widely distributed in all domains of life. In bacteria, CorC has been implicated in the survival of pathogenic microorganisms. In humans, CNNM proteins are involved in various biological events, such as body absorption/reabsorption of Mg(2+) and genetic disorders. This study determined the crystal structure of the Mg(2+)-bound CorC TM domain dimer. Each protomer has a single Mg(2+) binding site with a fully dehydrated Mg(2+) ion. The residues at the Mg(2+) binding site are strictly conserved in both human CNNM2 and CNNM4, and many of these residues are associated with genetic diseases. Furthermore, the structures were determined of the CorC cytoplasmic region containing its regulatory ATP-binding domain. A combination of structural and functional analyses not only revealed the potential interface between the TM and cytoplasmic domains but also showed that ATP binding is important for the Mg(2+) export activity of CorC (Huang, 2021a).

PRL3 phosphatase active site is required for binding the putative magnesium transporter CNNM3

The phosphatases of regenerating liver (PRLs) are involved in tumorigenesis and metastatic cancer yet their cellular function remains unclear. Recent reports have shown PRL phosphatases bind tightly to the CNNM family of membrane proteins to regulate magnesium efflux. This study characterize the interactions between the CBS-pair (Bateman) domain of CNNM3 and either PRL2 or PRL3 using X-ray crystallography, isothermal titration calorimetry, and activity assays. Four new crystal structures of PRL proteins bound to the CNNM3 CBS-pair domain are reported that reveal the effects of cysteine disulphide formation and nucleotide binding on complex formation. Comprehensive mutagenesis of the PRL3 catalytic site is reported to quantify the importance of different PRL amino acids, including cysteine 104, leucine 108, and arginine 110, for CNNM binding and phosphatase activity. The PRL3 R138E mutant is selectively deficient in CNNM3 binding with the potential to distinguish between the downstream effects of phosphatase and CNNM-binding activities in vivo. Through a novel activity assay, it was shown that PRL3 has magnesium-sensitive phosphatase activity with ATP and other nucleotides. These results identify a strong correlation between phosphatase activity and CNNM binding and support the contention that PRL function as pseudophosphatases regulated by chemical modifications of their catalytic cysteine (Zhang, 2017).

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Accogli, A., Scala, M., Calcagno, A., Napoli, F., Di Iorgi, N., Arrigo, S., Mancardi, M. M., Prato, G., Pisciotta, L., Nagel, M., Severino, M. and Capra, V. (2019). CNNM2 homozygous mutations cause severe refractory hypomagnesemia, epileptic encephalopathy and brain malformations. Eur J Med Genet 62(3): 198-203. PubMed ID: 30026055

Arjona, F. J., Chen, Y. X., Flik, G., Bindels, R. J. and Hoenderop, J. G. (2013). Tissue-specific expression and in vivo regulation of zebrafish orthologues of mammalian genes related to symptomatic hypomagnesemia. Pflugers Arch 465(10): 1409-1421. PubMed ID: 23636770

Chen, Y. S., Kozlov, G., Fakih, R., Funato, Y., Miki, H. and Gehring, K. (2018). The cyclic nucleotide-binding homology domain of the integral membrane protein CNNM mediates dimerization and is required for Mg(2+) efflux activity. J Biol Chem 293(52): 19998-20007. PubMed ID: 30341174

Coulthard, A. B., Alm, C., Cealiac, I., Sinclair, D. A., Honda, B. M., Rossi, F., Dimitri, P. and Hilliker, A. J. (2010). Essential loci in centromeric heterochromatin of Drosophila melanogaster. I: the right arm of chromosome 2. Genetics 185(2): 479-495. PubMed ID: 20382826

Guo, P., Xu, X., Wang, F., Yuan, X., Tu, Y., Zhang, B., Zheng, H., Yu, D., Ge, W., Gong, Z., Yang, X. and Xi, Y. (2019). A Novel Neuroprotective Role of Phosphatase of Regenerating Liver-1 against CO2 Stimulation in Drosophila. iScience 19: 291-302. PubMed ID: 31404830

Hirata, Y., Funato, Y. and Miki, H. (2014). Basolateral sorting of the Mg(2)(+) transporter CNNM4 requires interaction with AP-1A and AP-1B. Biochem Biophys Res Commun 455(3-4): 184-189. PubMed ID: 25449265

Huang, Y., Jin, F., Funato, Y., Xu, Z., Zhu, W., Wang, J., Sun, M., Zhao, Y., Yu, Y., Miki, H. and Hattori, M. (2021a). Structural basis for the Mg(2+) recognition and regulation of the CorC Mg(2+) transporter. Sci Adv 7(7). PubMed ID: 33568487

Huang, Y., Mu, K., Teng, X., Zhao, Y., Funato, Y., Miki, H., Zhu, W., Xu, Z. and Hattori, M. (2021b). Identification and mechanistic analysis of an inhibitor of the CorC Mg(2+) transporter. iScience 24(4): 102370. PubMed ID: 33912817

Ishii, T., Funato, Y., Hashizume, O., Yamazaki, D., Hirata, Y., Nishiwaki, K., Kono, N., Arai, H. and Miki, H. (2016). Mg2+ extrusion from intestinal epithelia by CNNM proteins is essential for gonadogenesis via AMPK-TORC1 signaling in Caenorhabditis elegans. PLoS Genet 12(8): e1006276. PubMed ID: 27564576

Urwyler, O., Izadifar, A., Vandenbogaerde, S., Sachse, S., Misbaer, A. and Schmucker, D. (2019). Branch-restricted localization of phosphatase Prl-1 specifies axonal synaptogenesis domains. Science 364(6439). PubMed ID: 31048465

Wu, Y., Funato, Y., Meschi, E., Jovanoski, K., Miki, H. and Waddell, S. (2020). Magnesium efflux from Drosophila Kenyon Cells is critical for normal and diet-enhanced long-term memory. Elife 9. PubMed ID: 33242000

Zhang, H., Kozlov, G., Li, X., Wu, H., Gulerez, I. and Gehring, K. (2017). PRL3 phosphatase active site is required for binding the putative magnesium transporter CNNM3. Sci Rep 7(1): 48. PubMed ID: 28246390

date revised: 5 May 2021

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