Lamin Mutations Accelerate Aging via Defective Export of Mitochondrial mRNAs through Nuclear Envelope Budding

Abstract

Defective RNA metabolism and transport are implicated in aging and degeneration [1, 2], but the underlying mechanisms remain poorly understood. A prevalent feature of aging is mitochondrial deterioration [3]. Here, we link a novel mechanism for RNA export through nuclear envelope (NE) budding [4, 5] that requires A-type lamin, an inner nuclear membrane-associated protein, to accelerated aging observed in Drosophila LaminC (LamC) mutations. These LamC mutations were modeled after A-lamin (LMNA) mutations causing progeroid syndromes (PSs) in humans. We identified mitochondrial assembly regulatory factor (Marf), a mitochondrial fusion factor (mitofusin), as well as other transcripts required for mitochondrial integrity and function, in a screen for RNAs that exit the nucleus through NE budding. PS-modeled LamC mutations induced premature aging in adult flight muscles, including decreased levels of specific mitochondrial protein transcripts (RNA) and progressive mitochondrial degradation. PS-modeled LamC mutations also induced the accelerated appearance of other phenotypes associated with aging, including a progressive accumulation of polyubiquitin aggregates [6, 7] and myofibril disorganization [8, 9]. Consistent with these observations, the mutants had progressive jumping and flight defects. Downregulating marf alone induced the above aging defects. Nevertheless, restoring marf was insufficient for rescuing the aging phenotypes in PS-modeled LamC mutations, as other mitochondrial RNAs are affected by inhibition of NE budding. Analysis of NE budding in dominant and recessive PS-modeled LamC mutations suggests a mechanism by which abnormal lamina organization prevents the egress of these RNAs via NE budding. These studies connect defects in RNA export through NE budding to progressive loss of mitochondrial integrity and premature aging.

Keywords: ATP synthase; Drosophila; Marf; RNA export; aging; mitochondria; mitochondrial assembly regulatory factor; nuclear envelope budding; progeroid syndrome (PS); sarcopenia.

Figure 1. Association of marf mRNA with DFz2C and nuclear LamC foci

(see also Figure S1) (A, C–G) FISH of a larval body wall muscle preparations using a (A) marf (C) ATP-syn-β, (D) blw, (E) ATP-syn-B, (F) cox4, (G) pink1 DNA probe and antibodies to LamC. Arrows denote foci containing mitochondrial transcripts. (B) Real time PCR of marf and mad after RNA-immunoprecipitation with DFz2C antibody from larval body wall muscles. (I) Schematic depiction of Drosophila LamC and human LMNA protein structure, indicating conserved (red vertical lines) and non-conserved (blue vertical lines) amino acid residues that when mutated cause progeroid syndromes in humans. Blue arrow indicates the mutation responsible for most HGPS cases. (H) Western blot of body wall muscle protein extracts from animals of the indicated genotypes probed with antibodies to LamC (top) and tubulin (Tub; bottom). Numbers at the right represent molecular weight in KDa. ***p<0.001 Error bars= SEM. Calibration bar in A, C–G is 6μm.

Figure 2. Age-dependent alterations in adult muscle mitochondria upon expressing LamC-E174K

(A–I, L) DLMs labeled with anti-ATP-syn-α and rhodamine-conjugated phalloidin from (A–C) 3- (D–F L) 14- and (G–I) 21-day old (A,D,G) MHC-Gal4 control; (B,E,H) LamC-WT, lamC/+; (C,F,I) LamC-E174K, lamC/+ and (L) LamC-E174K, lamC/+, Marf OE (J) Quantification of mitochondrial volume normalized to muscle volume. (K) Real time PCR of marf RNA levels in adult thoracic muscle from the indicated genotypes. N (left–right)= J: 30,49,28; 35,33,31; 28,47,32; 36. ***p< 0.001 Error bars= SEM. Calibration scale is 9μm.

Figure 3. Disruption of adult muscle mitochondria and progressive polyubiquitin aggregate accumulation upon expressing LamC-E174K

(See also Figures S2 and S3) (A–H) EM micrographs of adult DLMs showing mitochondrial cristae structure in (A) 60-day, (B,D,G) 14- and (C,E,F,H) 21-day old flies of the following genotypes: (A) MHC-Gal4/+, (B,C) LamC-E174K, lamC/+, (D–E) Marf-RNAi-muscle, (F) Blw-RNAi-muscle, (G) lamC-E174K, lamC/+ Marf OE, (H) DFz2-DN OE muscle. Arrowheads denote collapsing cristae; arrows denote disintegrating cristae. (I–N) DLMs double labeled anti-ubiquitin and Rhodamine-conjugated phalloidin in (I–J) 3-, (K–N) 14-day old adults from (I,K) LamC-WT, lamC/+; (J,L) LamC-E174K, lamC/+; (M) Marf-RNAi-muscle and (N) LamC-E174K, lamC/+ Marf OE. (O–P) Quantification of ubiquitin aggregate volume normalized to muscle volume. N (left to right)= O: 29,30,29; P:85,30,35,36,36. *p<0.05 ***p<0.001; Error bars= SEM Calibration bar is 0.5μm for (A,B,H), 0.4μm for (D,E,F,G) , 1μm for (C) and 16μm for (I–N)

Figure 4. Expression of PS-modeled mutations disrupts NE morphology and ultrastructure

(see also Figure S4) (A–B) Wing position in (A) control flies, showing normal wing position and (B) LamC-E174K, lamC/+ flies showing a “wings up” phenotype. (C) Quantification of landing height in 4- and 21-day old adults. (F–K) Larval body wall muscle nuclei labeled with anti-LamC or anti-LamDm0, and anti-DFz2C antibody from (F) BG487-Gal4/+ control; (G) LamC-WT, lamC/+; Arrows denote LamC/DFz2C foci; Arrowheads denote abnormal LamC blebs. (H) LamC-E174K, lamC/+; (I) lamC null mutant; (J) LamC-WT, lamC; and (K) LamC-r564c, lamC. Arrows point to DFz2C/LamC foci, and arrowheads to blebs. (D,E) Quantification of the number of DFz2C/LamC foci at body wall muscles normalized to BG487/+ controls in the indicated genotypes in a (D) lamC/+ or (E) lamC null mutant background. (L,M) Quantification of the percentage of body wall muscle nuclei containing more than 10 blebs at the NE in the indicated genotypes (L) in a lamC/+ heterozygous background and (M) in a lamC null mutant background. Control is BG487/+ in both graphs. (N–R) TEM of larval body wall muscle nuclei from (N) LamC-WT, lamC; (O–Q) LamC-E174K, lamC; and (R) LamC-r564c, lamC. White arrows denote megaRNP granules; White arrowheads denote empty blebs; Black arrows denote thickened lamina; Black arrowheads denote thickened lamina at the neck of blebs. *p<0.05 **p<0.01 ***p<0.001 Error bars= SEM N (left to right)= C: 81, 78, 83, 166, 83, 85, 89; D, E, L, M(number of hemisegments/number of nuclei) D: 21/1137,21/1026,24/1248; E:17/958,13/666,17/951,19/977; L: 20/1155, 20/1106, 20/1035, 12/647; M:26/1500,14/720,23/1274,19/1079,16/829. Calibration bar is 5.5μm for F–K, 1.4μm for P and 0.4μm for N, O, Q, R