Behavioral and Electrophysiological Characterization of Dyt1 Heterozygous Knockout Mice

DYT1 dystonia is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most of the patients have a trinucleotide deletion (ΔGAG) corresponding to a glutamic acid in the C-terminal region (torsinA^ΔE). Dyt1 ΔGAG heterozygous knock-in (KI) mice, which mimic ΔGAG mutation in the endogenous gene, exhibit motor deficits and deceased frequency of spontaneous excitatory post-synaptic currents (sEPSCs) and normal theta-burst-induced long-term potentiation (LTP) in the hippocampal CA1 region. Although Dyt1 KI mice show decreased hippocampal torsinA levels, it is not clear whether the decreased torsinA level itself affects the synaptic plasticity or torsinA^ΔE does it. To analyze the effect of partial torsinA loss on motor behaviors and synaptic transmission, Dyt1 heterozygous knock-out (KO) mice were examined as a model of a frame-shift DYT1 mutation in patients. Consistent with Dyt1 KI mice, Dyt1 heterozygous KO mice showed motor deficits in the beam-walking test. Dyt1 heterozygous KO mice showed decreased hippocampal torsinA levels lower than those in Dyt1 KI mice. Reduced sEPSCs and normal miniature excitatory post-synaptic currents (mEPSCs) were also observed in the acute hippocampal brain slices from Dyt1 heterozygous KO mice, suggesting that the partial loss of torsinA function in Dyt1 KI mice causes action potential-dependent neurotransmitter release deficits. On the other hand, Dyt1 heterozygous KO mice showed enhanced hippocampal LTP, normal input-output relations and paired pulse ratios in the extracellular field recordings. The results suggest that maintaining an appropriate torsinA level is important to sustain normal motor performance, synaptic transmission and plasticity. Developing therapeutics to restore a normal torsinA level may help to prevent and treat the symptoms in DYT1 dystonia.     

鼠肝炎冠状病毒非结构蛋白1及其突变体的原核表达

目的:构建鼠肝炎冠状病毒(MHV)非结构蛋白1(NSP1)及其突变体(NSP1 mu)的原核重组表达质粒,在大肠杆菌中分别融合表达重组NSP1及NSP1 mu。方法:以现有质粒载体为模板,扩增编码NSP1及NSP1 mu的基因片段,并克隆至pMD18-T克隆载体;菌落PCR鉴定阳性克隆并测序分析;将阳性克隆的目的片段亚克隆至表达载体pET-28a,并转化大肠杆菌TOP10感受态细胞,PCR和双酶切鉴定转化菌落;将阳性质粒转化大肠杆菌BL21(DE3)感受态细胞并加入IPTG诱导表达,SDS-PAGE和免疫印迹分析目的蛋白的表达。结果:PCR扩增得到表达NSP1及NSP1 mu的特异片段,并克隆到pMD18-T载体,测序结果正确无误;构建了NSP1和NSP1 mu的重组表达质粒,并在大肠杆菌BL21(DE3)中分别融合表达了重组NSP1及NSP1 mu,表达的目的蛋白均能与His单克隆抗体特异结合;用Ni-NTA琼脂糖试剂盒纯化重组蛋白,获得可溶性的NSP1及NSP1 mu,相对分子质量分别为27×103和28×103。结论:在大肠杆菌中分别表达并纯化获得了大量可溶性重组NSP1及NSP1 mu。     

人Polo样激酶1活性缺失突变体和结构域在293细胞中的瞬时表达

目的:构建人Polo样激酶1(Plk1)活性缺失突变体及结构域突变体的真核表达载体,并在293细胞中表达。方法:用二次PCR方法扩增Plk1基因并点突变,将82位赖氨酸突变为精氨酸,定向克隆到pcDNA3-Flag载体中;用普通PCR方法扩增Plk1激酶区域及Polo盒区域(PBD)基因,定向克隆到pcDNA3-Flag载体中;将上述质粒转染293细胞进行瞬时表达,Western印迹检测Plk1蛋白的表达。结果:构建了Flag-Plk1(K82R)、Flag-Plk1KD、Flag-Plk1PBD真核表达质粒,在293细胞中均可有效表达,蛋白相对分子质量分别为68×103、45×103、31×103。结论:在293细胞中表达了Flag-Plk1(K82R)、Flag-Plk1KD、Flag-Plk1PBD蛋白,有助于进一步探究Plk1对底物的功能。     

Pore Dynamics and Conductance of RyR1 Transmembrane Domain

Ryanodine receptors (RyR) are calcium release channels, playing a major role in the regulation of muscular contraction. Mutations in skeletal muscle RyR (RyR1) are associated with congenital diseases such as malignant hyperthermia and central core disease (CCD). The absence of high-resolution structures of RyR1 has limited our understanding of channel function and disease mechanisms at the molecular level. Previously, we have reported a hypothetical structure of the RyR1 pore-forming region, obtained by homology modeling and supported by mutational scans, electrophysiological measurements, and cryo-electron microscopy. Here, we utilize the expanded model encompassing six transmembrane helices to calculate the RyR1 pore region conductance, to analyze its structural stability, and to hypothesize the mechanism of the Ile4897 CCD-associated mutation. The calculated conductance of the wild-type RyR1 suggests that the proposed pore structure can sustain ion currents measured in single-channel experiments. We observe a stable pore structure on timescales of 0.2 μs, with multiple cations occupying the selectivity filter and cytosolic vestibule, but not the inner chamber. We further suggest that stability of the selectivity filter critically depends on the interactions between the I4897 residue and several hydrophobic residues of the neighboring subunit. Loss of these interactions in the case of polar substitution I4897T results in destabilization of the selectivity filter, a possible cause of the CCD-specific reduced Ca²⁺ conductance.     

Pore Dynamics and Conductance of RyR1 Transmembrane Domain

Ryanodine receptors (RyR) are calcium release channels, playing a major role in the regulation of muscular contraction. Mutations in skeletal muscle RyR (RyR1) are associated with congenital diseases such as malignant hyperthermia and central core disease (CCD). The absence of high-resolution structures of RyR1 has limited our understanding of channel function and disease mechanisms at the molecular level. Previously, we have reported a hypothetical structure of the RyR1 pore-forming region, obtained by homology modeling and supported by mutational scans, electrophysiological measurements, and cryo-electron microscopy. Here, we utilize the expanded model encompassing six transmembrane helices to calculate the RyR1 pore region conductance, to analyze its structural stability, and to hypothesize the mechanism of the Ile4897 CCD-associated mutation. The calculated conductance of the wild-type RyR1 suggests that the proposed pore structure can sustain ion currents measured in single-channel experiments. We observe a stable pore structure on timescales of 0.2 μs, with multiple cations occupying the selectivity filter and cytosolic vestibule, but not the inner chamber. We further suggest that stability of the selectivity filter critically depends on the interactions between the I4897 residue and several hydrophobic residues of the neighboring subunit. Loss of these interactions in the case of polar substitution I4897T results in destabilization of the selectivity filter, a possible cause of the CCD-specific reduced Ca²⁺ conductance.     

TBK1相关激酶活性缺失突变体和泛素样结构域突变体的构建、表达及生物活性鉴定

目的:构建TANK结合激酶1(TBK1)相关激酶活性缺失突变体和泛素样结构域突变体真核表达载体,检测该基因相关突变体在293细胞中的表达,并利用萤光素酶报告基因实验检测其生物活性。方法:根据文献报道的突变序列及QuickChange Site-Directed Mutagenesis实验设计手册,设计合成2条针对TBK1相关激酶活性缺失突变体和泛素样结构域突变体的引物,以实验室之前构建的TBK1野生型真核表达载体为模板,构建TBK1激酶活性缺失突变体和泛素样结构域突变体真核表达载体,分别命名为pcDNA3-Flag-TBK1(KD)、pcDNA3-Flag-TBK1(ΔULD)。以LipofactAMINE2000转染试剂转染至293细胞中进行瞬时表达,利用萤光素酶实验检测2种TBK1突变体诱导β干扰素(IFN-β)转录的情况。结果:测序结果表明,TBK1相关激酶活性缺失突变体和泛素样结构域缺失突变体真核表达载体构建成功,Western印迹检测表明其在293细胞中获得有效表达;用萤光素酶报告基因实验检测,与野生型TBK1相比,其相关激酶活性缺失突变体和泛素样结构域缺失突变体诱导IFN-β转录激活的作用明显降低。结论:真核表达的TBK1相关激酶活性缺失突变体和泛素样结构域突变体具有相应的生物学活性,为研究其功能奠定了基础。     

Effect of the Jimpy Mutation on Expression of Myelin Proteins in Heterozygous and Hemizygous Mouse Brain

The levels of myelin basic protein, proteolipid protein, and 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37) in cerebral hemispheres of wild-type, heterozygous jp/+, and hemizygous jp/Y mice of different ages were determined by radioimmunoassay and immunoblotting. In jp/Y brain the level of myelin basic protein was 8% that of wild-type at all ages. All forms of the protein were reduced although the 21.5K Mr form was relatively spared at early ages compared to the 18.5K, 17K, and 14K Mr forms. The level of 2',3'-cyclic nucleotide 3'-phosphohydrolase was 8% that of wild-type at all ages, and proteolipid protein was undetectable at any age. These results are consistent with the hypothesis that the jimpy mutation blocks myelin morphogenesis subsequent to incorporation of 21.5K Mr myelin basic protein but prior to incorporation of proteolipid protein. In jp/+ brain the levels of the three proteins were reduced commensurately to 60-70% those of wild-type. The deficit was apparent as early as 10 days after birth and remained proportionately constant throughout development. These results suggest that in jp/+ mice, X-chromosome inactivation produces a mosaic population of functionally wild-type and functionally jimpy oligodendrocytes. The former elaborate normal amounts of myelin but do not completely compensate for the myelin deficit due to the latter.     

Energetics of ErbB1 Transmembrane Domain Dimerization in Lipid Bilayers

One of the most extensively studied receptor tyrosine kinases is EGFR/ErbB1. Although our knowledge of the role of the extracellular domains and ligands in ErbB1 activation has increased dramatically based on solved domain structures, the exact mechanism of signal transduction across the membrane remains unknown. The transmembrane domains are expected to play an important role in the dimerization process, but the contribution of ErbB1 TM domain to dimer stability is not known, with published results contradicting one another. We address this controversy by showing that ErbB1 TM domain dimerizes in lipid bilayers and by calculating its contribution to stability as −2.5 kcal/mol. The stability calculations use two different methods based on Förster resonance energy transfer, which give the same result. The ErbB1 TM domain contribution to stability exceeds the change in receptor tyrosine kinases dimerization propensities that can convert normal signaling processes into pathogenic processes, and is thus likely important for biological function.     

A Unique Phenylalanine in the Transmembrane Domain Strengthens Homodimerization of the Syndecan-2 Transmembrane Domain and Functionally Regulates Syndecan-2

The syndecans are a type of cell surface adhesion receptor that initiates intracellular signaling events through receptor clustering mediated by their highly conserved transmembrane domains (TMDs). However, the exact function of the syndecan TMD is not yet fully understood. Here, we investigated the specific regulatory role of the syndecan-2 TMD. We found that syndecan-2 mutants in which the TMD had been replaced with that of syndecan-4 were defective in syndecan-2-mediated functions, suggesting that the TMD of syndecan-2 plays one or more specific roles. Interestingly, syndecan-2 has a stronger tendency to form sodium dodecyl sulfate (SDS)-resistant homodimers than syndecan-4. Our structural studies showed that a unique phenylalanine residue (Phe¹⁶⁷) enables an additional molecular interaction between the TMDs of the syndecan-2 homodimer. The presence of Phe¹⁶⁷ was correlated with a higher tendency toward oligomerization, and its replacement with isoleucine significantly reduced the SDS-resistant dimer formation and cellular functions of syndecan-2 (e.g. cell migration). Conversely, replacement of isoleucine with phenylalanine at this position in the syndecan-4 TMD rescued the defects observed in a mutant syndecan-2 harboring the syndecan-4 TMD. Taken together, these data suggest that Phe¹⁶⁷ in the TMD of syndecan-2 endows the protein with specific functions. Our work offers new insights into the signaling mediated by the TMD of syndecan family members.     

The Effect of the Shiverer Mutation on Myelin Basic Protein Expression in Homozygous and Heterozygous Mouse Brain

We report (a) that the shiverer mutation has pleiotropic phenotypic effects on myelin basic protein expression in the CNS of homozygous (shi/shi) mice and (b) that each of the effects of the shiverer allele is expressed co-dominantly with the wild-type allele in heterozygous (+/shi) animals. First, the total amount of myelin basic protein, as determined by radioimmunoassay, that accumulates in the CNS is approximately 0.1% of the wild-type amount in shi/shi animals and approximately 50% in +/shi animals. Second, the four major forms of myelin basic protein, with molecular weights of 21,500, 18,500, 17,000, and 14,000, that are present in wild-type mouse CNS are undetectable in either whole brain or purified myelin of shi/shi animals, and each of the four proteins is reduced commensurately in brain and myelin of +/shi animals. Third, the small amount of myelin basic protein-related material that does accumulate in the shi/shi brain consists of several polypeptides, with molecular weights ranging from 25,000 to 100,000, the pattern of which is different from that found in wild-type brain. The pattern of myelin basic protein-related polypeptides in +/shi brain is a composite of the wild type and the shiverer mutant. Fourth, messenger RNA from shi/shi brain, when translated in vitro, encodes a set of myelin basic protein-related polypeptides qualitatively similar to that encoded by wild-type messenger RNA, except that the 18,500 and 14,000 translation products are greatly reduced, while other myelin basic protein-related translation products are spared. The pattern of myelin basic protein-related translation products for +/shi messenger RNA is intermediate between the patterns for +/+ and shi/shi messenger RNAs. The results suggest that the genetic lesion in the shiverer mutation impinges on the structural gene (or genes) encoding myelin basic protein or on a cis-acting regulatory element controlling that gene (or genes).     

Alteration in Ion Channel Function of Mouse Nicotinic Acetylcholine Receptor by Mutations in the M4 Transmembrane Domain

The effect of structural alterations of the M4 transmembrane segment in the Torpedo californica AChR has shown that substitution of specific residues can be critical to the channel gating (Lasalde et al., 1996). In a previous study we found that phenylalanine and tryptophan substitutions at the αC418 residue in the M4 transmembrane segment of the Torpedo californica AChR significantly altered ion channel function (Lee et al., 1994; Ortiz-Miranda et al., 1997). Cassette mutagenesis was used to mutate the Cys residue at the corresponding C418 position in the α subunit of mouse AChR. A total of nine mutations on the mouse αC418 position were tested, including the αC418A, αC418V, αC418L, αC418S, αC418M, αC418W, αC418H, αC418E and αC418G mutants. All the mutants tested were functional except the αC418G which was not expressed on the surface of the oocyte. The data obtained from macroscopic and single channel currents demonstrate that different types of amino acids can be accommodated at this presumably lipid-exposed position without loss of ion-channel function. As with the Torpedo AChR, the mutation of Cys to Trp dramatically decreased the EC₅₀ for acetylcholine and increased channel open time. The lack of expression of the mouse αC418G suggest that there are some differences in folding, oligomerization and perhaps transport to the surface membrane for this mutant between the Torpedo and the mammalian AChR. Received: 30 December 1998/Revised: 13 April 1999     

Recombination Suppression by Heterozygous Robertsonian Chromosomes in the Mouse

Robertsonian chromosomes are metacentric chromosomes formed by the joining of two telocentric chromosomes at their centromere ends. Many Robertsonian chromosomes of the mouse suppress genetic recombination near the centromere when heterozygous. We have analyzed genetic recombination and meiotic pairing in mice heterozygous for Robertsonian chromosomes and genetic markers to determine (1) the reason for this recombination suppression and (2) whether there are any consistent rules to predict which Robertsonian chromosomes will suppress recombination. Meiotic pairing was analyzed using synaptonemal complex preparations. Our data provide evidence that the underlying mechanism of recombination suppression is mechanical interference in meiotic pairing between Robertsonian chromosomes and their telocentric partners. The fact that recombination suppression is specific to individual Robertsonian chromosomes suggests that the pairing delay is caused by minor structural differences between the Robertsonian chromosomes and their telocentric homologs and that these differences arise during Robertsonian formation. Further understanding of this pairing delay is important for mouse mapping studies. In 10 mouse chromosomes (3, 4, 5, 6, 8, 9, 10, 11, 15 and 19) the distances from the centromeres to first markers may still be underestimated because they have been determined using only Robertsonian chromosomes. Our control linkage studies using C-band (heterochromatin) markers for the centromeric region provide improved estimates for the centromere-to-first-locus distance in mouse chromosomes 1, 2 and 16.     

Transmembrane Helical Domain of the Cannabinoid CB1 Receptor

Brain cannabinoid (CB₁) receptors are G-protein coupled receptors and belong to the rhodopsin-like subfamily. A homology model of the inactive state of the CB₁ receptor was constructed using the x-ray structure of β₂-adrenergic receptor (β₂AR) as the template. We used 105 ns duration molecular-dynamics simulations of the CB₁ receptor embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer to gain some insight into the structure and function of the CB₁ receptor. As judged from the root mean-square deviations combined with the detailed structural analyses, the helical bundle of the CB₁ receptor appears to be fully converged in 50 ns of the simulation. The results reveal that the helical bundle structure of the CB₁ receptor maintains a topology quite similar to the x-ray structures of G-protein coupled receptors overall. It is also revealed that the CB₁ receptor is stabilized by the formation of extensive, water-mediated H-bond networks, aromatic stacking interactions, and receptor-lipid interactions within the helical core region. It is likely that these interactions, which are often specific to functional motifs, including the S(N)LAxAD, D(E)RY, CWxP, and NPxxY motifs, are the molecular constraints imposed on the inactive state of the CB₁ receptor. It appears that disruption of these specific interactions is necessary to release the molecular constraints to achieve a conformational change of the receptor suitable for G-protein activation.     

Gln-222 in Transmembrane Domain 4 and Gln-526 in Transmembrane Domain 9 Are Critical for Substrate Recognition in the Yeast High Affinity Glutathione Transporter, Hgt1p

Hgt1p, a member of the oligopeptide transporter family, is a high affinity glutathione transporter from the yeast Saccharomyces cerevisiae. We have explored the role of polar or charged residues in the putative transmembrane domains of Hgt1p to obtain insights into the structural features of Hgt1p that govern its substrate specificity. A total of 22 charged and polar residues in the predicted transmembrane domains and other conserved regions were subjected to alanine mutagenesis. Functional characterization of these 22 mutants identified 11 mutants which exhibited significant loss in functional activity. All 11 mutants except T114A had protein expression levels comparable with wild type, and all except E744A were proficient in trafficking to the cell surface. Kinetic analyses revealed differential contributions toward the functional activity of Hgt1p by these residues and identified Asn-124 in transmembrane domain 1 (TMD1), Gln-222 in TMD4, Gln-526 in TMD9, and Glu-544, Arg-554, and Lys-562 in the intracellular loop region 537–568 containing the highly conserved proline-rich motif to be essential for the transport activity of the protein. Furthermore, mutants Q222A and Q526A exhibited a nearly 4- and 8-fold increase in the K_m for glutathione. Interestingly, although Gln-222 is widely conserved among other functionally characterized oligopeptide transporter family members including those having a different substrate specificity, Gln-526 is present only in Hgt1p and Pgt1, the only two known high affinity glutathione transporters. These results provide the first insights into the substrate recognition residues of a high affinity glutathione transporter and on residues/helices involved in substrate translocation in the structurally uncharacterized oligopeptide transporter family.Hgt1p or ScOpt1p, a polytopic membrane protein, from the yeast Saccharomyces cerevisiae, was the first high affinity glutathione transporter to be identified in any system (1). Hgt1p belongs to a relatively novel family of transporters, the oligopeptide transporter (OPT)³ family, that contains a large number of fungal, plant, and prokaryotic members (2). The functional characterizations of a few of the fungal and plants members have demonstrated their ability to transport oligopeptides, glutathione, and metal-secondary amino acid conjugates by harnessing the proton gradient across the plasma membrane (3–7). Furthermore, these studies have also highlighted the physiological significance of this family in assimilation/mobilization of oligopeptides as nutrients in fungi and plants and in maintenance of metal homeostasis in plants. However, the majority of the members are yet uncharacterized and need to be defined with respect to their substrate specificity and physiological role.A complete lack of information on the structural features of the OPT family further limits our understanding of this large, uncharacterized family. Identification of residues or motifs critical for substrate recognition among the functionally characterized members would enable functional characterization of the new members within the family. This has prompted us to initiate a systematic study on the structure-function characterization of Hgt1p as a representative of the OPT family. Not only is Hgt1p the best characterized member of the OPT family in terms of its substrate specificity, being also able to transport some oligopeptides albeit with low affinity (1, 7, 8), its native host S. cerevisiae is a well established model system and easily amendable for mutagenesis-based structure-function studies. We have recently investigated the role of the 12 native cysteine residues in the structural stability and the transporter activity of the protein where 2 of the cysteines were found to be essential for functionality (9). However, no hints on the important motifs or conserved amino acids of Hgt1p (or any other member of the OPT family) that could be involved in substrate recognition have been obtained so far. In the current study we have focused on the polar and charged residues in the transmembrane domains of Hgt1p to explore their role in substrate recognition.Glutathione, the substrate for Hgt1p, is a hydrophilic substrate. Prior studies on structural characterization of transporters of the other hydrophilic substrates using biochemical and genetic strategies, such as site-directed mutagenesis and random mutagenesis, have established the role of polar and charged residues in the transmembrane domains of transporters in recognition, binding, and translocation of substrates (10–18). The availability of the crystal structures of a few transporter proteins have further enabled direct visualization of such interactions between the key residues in the transmembrane domains and the substrate molecule (19–22). In light of these studies we anticipated that few of the charged or polar residues in the predicted transmembrane domains of Hgt1p would be involved in substrate recognition and translocation across the membrane. Hence, a total of 22 polar or charged amino acids spanning the predicted transmembrane domains of Hgt1p were subjected to alanine scanning and functionally characterized. Detailed biochemical characterizations of these mutants revealed that Asn-124, Gln-222, Gln-526, Glu-544, Arg-554, and Lys-562 are key residues for the transport activity of Hgt1p. As replacement of Gln-222 in TMD4 and Gln-526 in TMD9 with alanine resulted in a significant decrease in the affinity of the transporter for glutathione, it suggested that the two residues might directly participate in glutathione recognition as a substrate. These observations provide the first insights into substrate binding residues in Hgt1p, a member of a novel and important transporter family (OPT family).     

FGFR3 Transmembrane Domain Interactions Persist in the Presence of Its Extracellular Domain

Isolated receptor tyrosine kinase transmembrane (TM) domains have been shown to form sequence-specific dimers in membranes. Yet, it is not clear whether studies of isolated TM domains yield knowledge that is relevant to full-length receptors or whether the large glycosylated extracellular domains alter the interactions between the TM helices. Here, we address this question by quantifying the effect of the pathogenic A391E TM domain mutation on the stability of the fibroblast growth factor receptor 3 dimer in the presence of the extracellular domain and comparing these results to the case of the isolated TM fibroblast growth factor receptor 3 domains. We perform the measurements in plasma membrane-derived vesicles using a Förster-resonance-energy-transfer-based method. The effect of the mutation on dimer stability in both cases is the same (∼−1.5 kcal/mol), suggesting that the interactions observed in simple TM-peptide model systems are relevant in a biological context.     

Transmembrane and Coiled-Coil Domain Family 1 Is a Novel Protein of the Endoplasmic Reticulum

The endoplasmic reticulum (ER) is a continuous membrane network in eukaryotic cells comprising the nuclear envelope, the rough ER, and the smooth ER. The ER has multiple critical functions and a characteristic structure. In this study, we identified a new protein of the ER, TMCC1 (transmembrane and coiled-coil domain family 1). The TMCC family consists of at least 3 putative proteins (TMCC1–3) that are conserved from nematode to human. We show that TMCC1 is an ER protein that is expressed in diverse human cell lines. TMCC1 contains 2 adjacent transmembrane domains near the C-terminus, in addition to coiled-coil domains. TMCC1 was targeted to the rough ER through the transmembrane domains, whereas the N-terminal region and C-terminal tail of TMCC1 were found to reside in the cytoplasm. Moreover, the cytosolic region of TMCC1 formed homo- or hetero-dimers or oligomers with other TMCC proteins and interacted with ribosomal proteins. Notably, overexpression of TMCC1 or its transmembrane domains caused defects in ER morphology. Our results suggest roles of TMCC1 in ER organization.     

Heterozygous Mutation of Drosophila Opa1 Causes the Development of Multiple Organ Abnormalities in an Age-Dependent and Organ-Specific Manner

Optic Atrophy 1 (OPA1) is a ubiquitously expressed dynamin-like GTPase in the inner mitochondrial membrane. It plays important roles in mitochondrial fusion, apoptosis, reactive oxygen species (ROS) and ATP production. Mutations of OPA1 result in autosomal dominant optic atrophy (DOA). The molecular mechanisms by which link OPA1 mutations and DOA are not fully understood. Recently, we created a Drosophila model to study the pathogenesis of optic atrophy. Heterozygous mutation of Drosophila OPA1 (dOpa1) by P-element insertion results in no obvious morphological abnormalities, whereas homozygous mutation is embryonic lethal. In eye-specific somatic clones, homozygous mutation of dOpa1 causes rough (mispatterning) and glossy (decreased lens deposition) eye phenotypes in adult Drosophila. In humans, heterozygous mutations in OPA1 have been associated with mitochondrial dysfunction, which is predicted to affect multiple organs. In this study, we demonstrated that heterozygous dOpa1 mutation perturbs the visual function and an ERG profile of the Drosophila compound eye. We independently showed that antioxidants delayed the onset of mutant phenotypes in ERG and improved larval vision function in phototaxis assay. Furthermore, heterozygous dOpa1 mutation also caused decreased heart rate, increased heart arrhythmia, and poor tolerance to stress induced by electrical pacing. However, antioxidants had no effects on the dysfunctional heart of heterozygous dOpa1 mutants. Under stress, heterozygous dOpa1 mutations caused reduced escape response, suggesting abnormal function of the skeletal muscles. Our results suggest that heterozygous mutation of dOpa1 shows organ-specific pathogenesis and is associated with multiple organ abnormalities in an age-dependent and organ-specific manner.