Introduction
Endoplasmic reticulum (ER)-mitochondria contact sites (ERMCSs) are crucial for multiple cellular processes such as calcium signaling, lipid transport, and mitochondrial dynamics. The molecular organization, functions, regulation of ERMCS, and the physiological roles of altered ERMCSs are not fully understood in higher eukaryotes. This article explores the role of Mitoguardin (Miga), a mitochondrial protein, in ERMCS formation and its implications for neurodegeneration, weight loss, and metabolic disorders.
The Significance of ER-Mitochondria Contact Sites
Intracellular organelles with membranes are characteristic features of eukaryotic cells. The compartmentalization of the cytoplasm enables specific cellular activities occur in restricted regions. The physical contacts between different organelles at defined loci allow both material and information exchange between organelles. ERMCSs play various roles including phospholipid exchange and calcium flux between ER and mitochondria. ERMCSs also function as platforms for mitochondrial division, coupling mitochondrial DNA synthesis with mitochondrial fission, autophagosome formation, and mitophagy.
Miga: A Key Regulator of ERMCS Formation
Our previous study identified a family of mitochondrial proteins called Mitoguardin (Miga). Miga contains a function unknown domain that is highly conserved from Caenorhabditis elegans to humans. Miga localizes on the outer membrane of mitochondria and promotes mitochondrial fusion through interaction with mitoPLD, a divergent family member of the phospholipase D family that is required for the fusion of mitochondrial outer membranes. Loss of Miga leads to fragmented mitochondria and reduced mitochondrial functions. Flies lacking Miga do not grow beyond the early pupal stage. The mosaic eyes in flies with Miga mutant clones degenerate with aging. Mammals have two Miga proteins: MIGA1 and MIGA2.
We found that Miga interacts with an ER protein Vap33 through its FFAT-like motif and an amyotrophic lateral sclerosis (ALS) disease related Vap33 mutation considerably reduces its interaction with Miga. Multiple serine residues inside and near the Miga FFAT motif were phosphorylated, which is required for its interaction with Vap33 and Miga-mediated ERMCS formation. The interaction between Vap33 and Miga promoted further phosphorylation of upstream serine/threonine clusters, which fine-tuned Miga activity. Protein kinases CKI and CaMKII contribute to Miga hyperphosphorylation. MIGA2, encoded by the miga mammalian ortholog, has conserved functions in mammalian cells.
Miga Overexpression and Neurodegeneration
Our previous studies indicated that a mitochondrial outer membrane protein Miga is required for neuronal homeostasis. Interestingly, we found that Miga overexpression led to severe retinal degeneration in fly eyes. We used GMR-Gal4, a Gal4 driver expressing in the developing eyes, to drive UAS-Miga expression and examined the adult fly eyes at days 1 and 30. The adult eyes with Miga overexpression looked grossly normal from outside. TEM analysis was performed for the retina thin sections of young (1 Day) and old (30 Day) flies with indicated genotypes. The ommatidia of the control (CTL) flies showed seven photoreceptor cells with intact rhabdomeres at both the young and old stages. GMR-Gal4 driven Miga overexpression resulted in reduction of rhabdomere number and size in the 1-day-old flies and the loss of photoreceptor cells in 30-day-old flies. Large amount of circular membrane structures accumulated in the photoreceptor cells. ER tubules attached to the mitochondria were increased in the photoreceptor cells. GMR-Gal4-driven Marf overexpression did not change the number and size of rhabdomeres in the 1-day-old flies, but slightly reduced the number and size of rhabdomeres in the 30-day-old animals. GMR-Gal4-driven MitoPLD overexpression did not affect the number and size of rhabdomeres in both 1-day and 30-day-old animals.
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We wondered whether the retinal degeneration of the Miga overexpressing eyes was due to the increased mitochondrial fusion. Therefore, we overexpressed Marf, the Drosophila mitofusin, and MitoPLD in a same manner as we overexpressed Miga. Surprisingly, neither Marf nor MitoPLD overexpression reduced the rhabdomere numbers or sizes in 1-day-old flies. We carefully examined the retina in 1-day-old flies and saw many circular shaped membrane structures inside the photoreceptor cells. Some of the circular, membraned structures were found to be donut-shaped mitochondria with closely attached ER tubules. There were also regular shaped mitochondria with close contacts with ER, which were seldom seen in wild-type control eyes. The proximity between ER and mitochondria at the contact sites were close to 10 nm and the ribosomes were excluded. Although several studies suggested that MFN2, the mammalian Marf homolog, mediated ERMCSs, we did not observe any increase in ERMCS when Marf was overexpressed in the fly eyes. MitoPLD overexpression did not affect ERMCSs either.
Miga's Interaction with Vap33
To understand Miga function, we performed tandem immunoprecipitation (IP) in the cultured S2 cells with FLAG-HA tandem tagged overexpressed Miga and examined its binding partner with mass spectrometry. The ER protein Vap33 was one of the binding partners of Miga. The mammalian Vap33 orthologs are VAPA and VAPB. Both proteins are ER proteins mediating contacts between ER and other organelles. Point mutation in VAPB has been identified in amyotrophic lateral sclerosis (ALS) patients. Recent study in mammalian cells found that MIGA2 form a complex with VAP proteins. We confirmed the interaction between Vap33 and Miga by IP. Miga and Vap33 could pull down each other in both directions. Miga-V5 and Vap33-Flag could pull down each other in both directions in the IP assay when both were overexpressed in S2 cells. The affinity between Miga-V5 and Vap33P58S-Flag was reduced compared with that between Miga-V5 and wildtype Vap33-Flag.
VAP proteins interact with its partner through an FFAT motif EFFDAXE. Miga has a conserved sequence that is similar to the FFAT motif but with two acidic amino acids changed to serine. We prepared an FFAT motif mutant form of Miga (MigaFM) with the 247th phenylalanine and the 249th serine residues changed to alanine. MigaFM failed to bind to Vap33. We expressed a genomic rescue construct of Miga that contains the genomic fragment of Miga and a 3 × HA tag fused at the C-terminus just before the stop codon of Miga and a Flag tagged Vap33 in S2 cells and performed IP experiments. Since the genomic rescue construct of Miga contains the regulatory sequences from Miga locus, it likely expresses Miga in an endogenous level. Indeed, the level Miga-HA was much less than the level of overexpressed ones. We found that Miga and Vap33 could pull down each other in this condition. We expressed both wildtype and mutant Miga with Vap33 in fly fat body tissues.
Role of Miga in Weight Loss and Metabolic Diseases
Mice with single or double knockout (KO) of Miga1 and Miga2 are viable. Both Miga2 and Miga1/2 KO mice showed reduced body weight and body fat. Under high-fat diet consumption, Miga2 KO mice has minimal lipidosis. In a combined association study, an SNP located in Miga1 was shown to be associated with subscapular skin-fold thickness in humans and back fat thickness in pigs. In addition, the female Miga1/2 KO mice were shown to be sub-fertile with reduced ovarian activity, oocyte quality, and embryo developmental potentials. Miga2 KO mice also has immune defects and showed severe depression. These findings suggest a potential role for Miga in regulating body weight and metabolism.
Although ERMCSs changes have been reported in Alzheimer’s disease and metabolic diseases such as obesity, there is no direct evidence indicating that defective ERMCSs led to disease conditions.
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Mirabegron and its effects
Mirabegron, the active substance in Betmiga, is a beta-3-adrenergic-receptor agonist. It works by attaching to and activating beta-3 receptors that are found in the muscle cells of the bladder. Experimental studies have shown that, when activated, beta-3 receptors cause the bladder muscles to relax. This is thought to lead to an increase in the capacity of the bladder and changes in the way the bladder contracts, resulting in fewer bladder contractions and thus fewer unwanted urinations.
Treatment with 50 mg a day of Betmiga was shown to be effective in reducing the number of urination and incontinence episodes. After 3 months of treatment, on average Betmiga 50 mg reduced the number of urinations by 1.8 per day compared with a reduction of 1.2 per day for placebo. Betmiga 50 mg resulted in a reduction of 1.5 incontinence episodes per day compared with a reduction of 1.1 incontinence episodes per day for placebo.
The most common side effects with Betmiga are tachycardia (rapid heartbeat) seen in just over 1 person in 100, and urinary tract infection (infection of the structures that carry urine) seen in just under 3 people in 100. Serious but uncommon side effects include atrial fibrillation (cardiac rhythm disorder).
Betmiga must not be used in people who have hypertension (high blood pressure) that is severe and uncontrolled.
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