建立第22型脊隨小腦萎縮症的果蠅模式

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(1)國立臺灣師範大學生命科學系碩士論文. 建立第 22 型脊髓小腦萎縮症的果蠅模式 Generating Drosophila Models for Spinocerebellar Ataxia Type 22. 研究生：林勁欣 Ching-Hsin Lin. 指導教授：蘇銘燦博士 Dr. Ming-Tsan Su. 中華民國 105 年 1 月. 0.

(2) Table of Content 中文摘要……………………………………………………….2. Abstract…………………………………………………………3. Introduction……………………………………………………..4. The objectives of study...……………………………………….8. Materials and Methods………………………………………...9. Result………………………………………………………….12. Discussion…………………………………………………..…16. Reference……………………………………………………...17. Figures………………………………………………………...22. 1.

(3) 中文摘要脊髓小腦共濟失調症（Spinocerebellar Ataxia，SCA）為一種顯性遺傳性的神經系統疾病，若雙親其中一位患有此症，其子代不分性別均有一半的罹患機率；雖是同一家族，其發病年齡和病徵也不盡相同。台北榮總與陽明大學從一家系共四代的台灣病人，經臨床鑑定確認患有顯性遺傳的小腦運動失調症，透過進行鏈鎖分析發現不同於已知的 SCA 亞型致病基因位點，且基因 CAG、CTG、 ATTCT 並無異常重複突變，判斷此為第 22 型脊髓小腦共濟失調症。通過全基因組鏈鎖分析定位出 SCA22 突變位於 1 號染色體 1p21-q23，基因變異是發生在編碼的鉀離子（voltage-gated potassium，Kv）通道 Kv4.3 的 KCND3 基因。其中在台灣及法國家族皆發現的基因變異為第 227 個胺基酸 Phenylalanine 缺失（p. ∆F227）；在美國猶太人及日本家族發現的則是第 345 個胺基酸由 Glycine 點突變成 Valine （p.G345V）。本研究的主要目的即為建立第 22 型脊髓小腦共濟失調症的果蠅模式株，利用過量表現 KCND3 突變基因，來探討基因變異造成的 Kv4.3 鉀離子通道蛋白對病理症狀如運動能力及壽命的影響，並釐清 SCA22 的致病機制。在本研究中，我們建立的 SCA22 模式果蠅，確實出現年齡伴隨的病理特徵，包括細胞凋亡、運動能力下降以及縮短壽命等退化症狀。此外，在 mRNA 及蛋白質表現量的測量中，發現∆F227 在蛋白質的轉譯功能可能發生異常，由於前人從免疫螢光分析的實驗，發現 p. ∆F227 的 Kv4.3 鉀離子通道蛋白無法正常上到細胞膜，且大部分保留在內質網（endoplasmic reticulum， ER），前人研究指出蛋白質累積在內質網會造成內質網壓力（ER stress），我們將近一步觀察細胞是否發生為因應此壓力而產生之相關蛋白質反應。此外由於研究指出鉀離子在調控細胞質離子的恆定上扮演極重要的角色，藉由細胞膜上的鈉鉀離子幫浦（Na+/K+-ATPase）將鉀離子主動運輸至細胞內，及鉀離子通道的開閉調控鉀離子外流，兩者作用平衡可維持細胞容積和防止細胞凋亡發生，我們也將進一步研究過表現 G345V 基因是否造成細胞內部離子失衡導致細胞的死亡。. 關鍵字：果蠅；脊髓小腦萎縮症 2.

(4) Abstract. The spinocerebellar ataxias (SCA) are a diverse group of autosomal dominant neurological disorders characterized by progressive degeneration of many nevrse systems, including cerebellum, spinocerebellar tracts, and brain stem neurons. Recent discovery of mutations in the voltage-gated potassium channel Kv4.3-encoded gene KCND3 has shown to be the cause of the autosomal dominant spinocerebellar ataxia type 22 (SCA22). Of all KCND3 mutations, the in-frame three-nucleotide deletion c.679_681delTTC p.F227del (KCND3-ΔF227) has been identified in either the French and Chinese pedigrees. The in-frame point mutation c.1304G>T p.G345V (KCND3-G345V) has been identified in either the American and Japanese pedigrees. Since the underlying pathomechanisms of SCA22 is poorly understood, we generate Drosophila models for SCA22 by overexpression of wild-type KCND3, and mutant KCND3 variants (i.e. ΔF227; G345V) using the UAS/Gal4 system to address the above question. Ectopic expression of mutant KCND3 cause various pathological features, including neurodegeneration, apoptosis, mobility defects and shortened lifespan. More detailed analysis of mRNA and protein expression level found that ΔF227 translation abnormally by decreases in protein production. Since immunocytochemistry analyses revealed that KCND3-ΔF227 retained in the endoplasmic reticulum (ER). We suspect that KCND3-ΔF227 might induces ER stress thereby inducing neurodegenerations. Additionally, KCND3 is a potassium channel, ectopic KCND3 expression may cause the imbalance intracellular potassium concentration and lead to neuronal cell death. All the above mentioned possible pathomechanisms will be investigated with the newly established models.. Key words: spinocerebellar ataxias, neurodegeneration, voltage-gated potassium channel Kv4.3, Drosophila, endoplasmic reticulumstress, K+ efflux 3.

(5) Introduction. Spinocerebellar ataxia type 22 Cerebellum plays an important role in human central nervous system (CNS). Cerebellum is responsible for integrating neural information to coordinate movements and to participate in motor planning. When the cerebellum or its direct connections are damaged, a wide range of characteristics clinical symptoms including poor body balance and disturbance in gait and posture arise. The spinocerebellar ataxia (SCA) is a group of neurodegenerative diseases, which leads to late-onset progressive cerebellum dysfunction and severe neuronal loss [1]. . Recently, many SCA-causing genes have been identified and under rigorously. studied. Ataxia is a nonspecific clinical manifestation implying dysfunction of the parts of the nervous system that coordinate movement. Several possible causes exist for these patterns of neurological dysfunction. Although in the same family, the patient may show different symptoms and different age of onset. With the difficult to clinical diagnosis based on the variety of symptoms, classification using molecular genetic analyses is important. The specific genetic loci for SCA1, SCA2, SCA3, SCA4, SCA5, SCA6, SCA7, SCA8, SCA10, SCA11, SCA12, SCA13, SCA14, SCA16, SCA17, and dentatorubral pallidoluysian atrophy (DRPLA) have been revealed [2]. Most of the abovementioned diseases have shown to be caused by the expansion of repeat nucleotides (i.e. CAG, CTG, ATTCT) [3-14]. In an effort to identify the disease-causing gene for SCA22, the researchers from Veterans General Hospital and of Taiwan and Yang-Ming University have focused on a four-generation Taiwanese family in which the cerebellar ataxia segregates in an autosomal dominant mode [15]. Since sequence analyses have failed to identify any mutation from 16 known SCA-causing genes. Besides, no common disease-causing repeat nucleotide expansions (i.e. CAG, CTG, ATTCT) were found, suggesting that 4.

(6) novel genes are involved in SCA22. Through pedigree analysis, they have mapped the corresponding locus to chromosome 1p21-q23. Further mutation analyses from other SCA22 families, such as French, Ashkenazi Jewish and Japanese, demonstrated that the SCA22 is caused by the mutations in the voltage-gated potassium channel Kv4.3-encoding gene KCND3 [16].. Voltage-gated potassium channel Kv4.3 The voltage-gated potassium (Kv) channel is one of the most diverse ion channel families. The voltage-gated potassium (Kv) channels of eukaryotes are involved in neural signaling, generation of the cardiac rhythm, shaping the action potential, neuronal excitability and plasticity [17-19]. These are all highly similar proteins composed of two subunits, a large catalytic subunit (alpha) and a smaller glycoprotein subunit (beta). With few small amino acid substitutions in the potassium channel, it changes the diversity of the voltage-dependent gates mechanism, channel conductance and toxin binding properties. On the basis of sequence similarity and function, these Kv families can be divided into several subfamilies: Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). Each type of potassium channel consists of pore-forming alpha subunits which associate with different types of beta subunit [20, 21]. Based on amino acid sequence homology, Drosophila Shal protein is the Kv4 family. These channels are the primary subunits contributing to transient, voltage-dependent potassium currents in the nervous system (A currents) and the heart (transient outward current), which can be inhibited by free fatty acids [22]. The Kv4 family can be further divided into 3 subfamilies, designated Kv4.1 (KCND1), Kv4.2 (KCND2) and Kv4.3 (KCND3). Kv4.3 channels are important molecular components of transient K+ currents in brain and heart. Two isoforms of Kv4.3 have been cloned: one is full length and the short form consists of small amino acid deletion. Either 5.

(7) forms are expressed in the brain, whereas the longer isoform is expressed in the heart only [23].. ER stress Previous studies revealed that the expression of KCND3-ΔF227 mutant protein is fewer than wild-type KCND3 and mutant KCND-G345V when transfected transiently in HEK293 cells with immunocytochemistry analyses. Additionally, KCND3-ΔF227 mutant protein did not move to the plasma membrane and retained in the endoplasmic reticulum (ER) [16]. In eukaryotic cells, ER plays an important role in lipid synthesis, protein folding and protein maturation. Recently, endogenous cellular stress, such as ER stress, have been identified as a cause of cell death. For instance, the accumulation unfolded proteins within a cell's ER leads to stress and aberrant homeostasis. The conditions interfering with the function of ER are collectively called ER stress. In order for cell to survival, cells initiates a series of signaling pathways, including unfolded-protein response (UPR), to counterbalance this type of stress [24, 25]. The UPR signaling pathway could reduce the number of unfolded proteins in the ER through an efficient mechanism by the phosphorylating the α-subunit of eukaryotic initiation factor 2 (eIF2) which inhibits protein translation [26]. The UPR signaling also activates downstream effectors of activating transcription factor 6 (ATF6) and inositol-requiring protein-1 (IRE1) through up-regulating the expression level of ER-resident chaperones, including GRP78/BiP and GRP94. GRP78/BiP and GRP94 can increases the folding capacity of the ER, providing a protective effect for cell survival [27-29]. If the stress stimulus is unresolved, then cells activate death signaling pathways [30].. Potassium efflux Several studies suggest the disruption of ion homeostasis has associated with a number of neurodegenerations[31]. The link between ion and the pathogenesis of 6.

(8) several neurodegenerative was found to mediate through the changes in intracellular ion homeostasis. The aberrant ion homeostasis commonly induces apoptosis and loss of neurons [32, 33]. As K+ is the most abundant iron in the cytoplasm, cellular apoptosis is strongly influenced by intracellular K+ levels [34]. Na+/K+ -ATPase is an integral membrane protein responsible for establishing and maintaining the charge difference of Na+ and K+ across the plasma the membrane and the osmotic balance of animal cells [35-38]. Since the Kv channel activity gives rise to transient outward K+ currents [39]. . The balance between Na+/K+ -ATPase and Kv channel is important for. maintaining intracellular K+ hemostasis and cell volume [40, 41]. K+ efflux, K+ loss from the intracellular space, have been proposed to be a key early stage in induction of apoptosis [42-46] and apoptotic cell body shrinkage [47].. 7.

(9) The objectives of study. Drosophila melanogaster (fruit fly) have been emerged as an excellent model organism to study the interactions between aging and neurodegenerative diseases. This is due to the facts that the lifespan of Drosophila is short and Drosophila has well-differentiated brain and nervous system much like mammalian [48]. Particularly, the genetic circuits that control Drosophila behavior ranging from simple avoidance to learning and memory [49] are well-established. With its long history in biological research, the fully genome sequences have been well studied and are available for genetic manipulation [50]. In the present study, we aimed to generate transgenic Drosophila models for SCA22 by overexpression of wild-type KCND3 and mutant KCND3-(ΔF227; G345V). The established SCA22 fly models will be further characterized and used for dissecting the underlying pathomechanisms of SCA22. For the generation of transgenic fly models, the wild type human Kv4.3-encoded gene KCND3 and mutant variants (∆F227, G345V) were cloned by phiC31 site-specific germ-line transformation vectors. Transgenic flies were obtained through microinjection. The expression of transgenes were under the control of the GAL4/UAS system. Upon the overexpression of KCND3 transgenes, we examined various neurodegeneration phenotypes, including shorten lifespan, defect of climbing ability and brain structure degeneration. Also, with the driver of drosophila inhibitor of apoptosis, gmr>DIAP2, alleviate the neurodegeneration phenotype in retinal cells. Demonstrate that age-dependent apoptosis does participate in SCA22 pathogenesis mechanism.. 8.

(10) Materials and Methods. Drosophila stocks and transgenic flies All Drosophila melanogaster stocks were raised and the genetic experiments were conducted at 25°C or 29°C on standard cornmeal-yeast-agar medium supplied by Institute of Molecular Biology Academia Sinica. We anaesthetize flies with CO2 then observe and manipulate them with a stereomicroscope. Most Drosophila stocks were obtained from the Bloomington stock center. The wild type and mutant human kv4.3 and cDNAs were gifts from Taipei Veterans General Hospital. Each cDNA fragment was cloned into pBID-UASC vector using In-Fusion HD cloning kit (clontech). The transgenic fly lines were produced through phiC31 integrase system (Fig. 1). Then the plasmid was site-directed injected into Drosophila embryos by GENETIC SERVICES, INC. All transgenes were backcrossed into the w genetic background and balanced with Cyo chromosome.. USA-GAL4 system The ectopic expression of transgenes were achieved using the UAS/Gal4 system [51]. Male flies carrying UAS-KCND3, UAS-KCND3-∆F227, or UAS-KCND3-G345V expression constructs were crossed to female flies with the neuronal elav-GAL4 driver on the third chromosome. Retinal overexpression of transgenes were achieved using photoreceptor driver GMR-GAL4 on the second chromosome. The Drosophila inhibitor of apoptosis (IAP) were examined using P{GMR>DIAP2} on the third chromosome. Male F1 progeny carried either UAS or GAL4 constructs and were used for subsequent analyses. Each heterogeneous Gal4 driver and UAS lines was used as control.. 9.

(11) Western blotting For protein extraction, 15 heads from transgenic flies were homogenized in 2× Laemmli sample buffer containing β-mercaptoethanol and boiled at 100oC for 5 min. Proteins were separated on 10% sodium dodecyl sulfate–polyacrylamide (SDS–PAGE) gels at 150 V for 1 h. Gels were then transferred to nitrocellulose membranes, and incubated in a blocking solution containing 5% milk in TBST overnight at 4 °C, then probed with primary antibodies for 1 h at room temperature. The following primary antibodies used: anti-Kv4.3 polyclonal antibody which recognizes all transgene line, monoclonal anti-ß-actin was used as loading control in each sample.. RNA extraction, reverse transcription PCR and real time PCR Total RNA was extracted in Trizol (Invitrogen) from the heads of 1-day-old male flies carrying transgene driven by elav-GAL4 using the RNeasy Kit (Qiagen) and was then used for reverse transcription with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The quantities of cDNA were used in Taqman gene expression assay on an StepOnePlus Real-time PCR system (Applied Biosystems) depended on the expression of gene. the probe KCND3 (assay ID AJHSOHX), Actin (assay ID Dm02371594_s1) were used. Each set of experiments was performed in duplicated and was repeated at least three times.. Climbing ability and analysis The climbing assays were performed as previously described [52]. Briefly, The climbing apparatus is a 30 cm long glass tube, with a diameter of 15 mm. the tube is held in place by a plastic funnel as a means for transferring flies intp the apparatus and acts as either a base for tube. The glass tube (30 cm long with a diameter of 15 mm) is divided into a series of five section, starting from the base, each 2 cm in height (scored 1-5), with an buffer zone in the upper portion of the apparatus. Flies were allowed 10 seconds to climb after being tapped down and are given a score 10.

(12) based upon the sections reached. The flies are scored ten times (trials) per climbing section, from which a climbing index is calculated. For each assay, thirty flies from each genotype with different age were used.. Lifespan analysis Flies were raised to adulthood and newly eclosed flies were placed in vials at low density (10-25 flies per vial) incubated at 25°C or 29°C. Flies were transferred to fresh food vials every 5 day, and dead flies were scored. The difference in lifespan was tested using the log-rank test.. Paraffin Histology Male fly heads were fixed 15 minutes in 4% paraformaldehyde and dehydrated using an ethanol-xylene series each step steep 5 minutes. Sliced the paraffin-embedded head into section of section (5 μm) using a microtome (Leica RM2135). Then collect the section stained with hematoxylin and eosin (H&E) and visualize under microscope (Leitz wetzlar orthoplan microscope).. Acridine Orange Staining The stain method were performed as previously described [53]. Freshly made 5 μg/mL acridine orange (Sigma, MO) stain and stored in a dark container at 4°C. Male fly heads were dissected in PBS (phosphate-buffered saline) and the wings and legs are removed. Using one set of forceps to pinch the proboscis, a small section of the back of the head containing no brain tissue is grasped. Carefully remove tissue debris and save the whole brain. Once isolated, the brain is transferred from the PBS onto a slide with a p200 micropipette tip along with ~20 μL of PBS. The PBS is removed from the slide and replaced with 30 μL of 5 μg/mL acridine orange. Incubate fly brains 5 minutes in AO solution. Mount Paraffin oil and view with a cover slip under confocal laser scanning microscopy. 11.

(13) Results Generation of transgenic SCA22 fly models We have obtained several transgenic flies carrying UAS-KCND3, UAS-KCND3-∆F227 and UAS-KCND3-G345V expression constructs (Fig. 1). We first test the expression of these transgenes driven by GMR-GAL4. Real-time PCR analysis showed that all transgene flies express KCND3 mRNA in comparable level, but not in GMR-GAL4 control flies (Fig. 2A). Western blotting analysis showed that either wild type or mutant Kv4.3 proteins were expressed in the heads of transgenic flies, nor in GMR-GAL4 control flies (Fig. 2B). The expression of wild type and G345V mutant proteins were compatible. However, the expression of KCND3-ΔF227 protein was considerably less than those of wild type and G345V mutant proteins. Characterization of SCA22 fly models Since SCA22 is dominantly inherited, we have overexpressed either wild type or mutant KCND3 in transgenic flies using various Gal4 drivers. For fast phenotypic assessments of age-dependent degeneration in fly models, compound eyes of flies were chosen because of their anatomical features which provide an easy assessment. To test if ectopic KCND3 expression would cause neurodegeneration, we showed that pan-neuronal overexpression of either wild type or mutant KCND3 under the control of elav-GAL4. In survivorship assay, the lifespan of elav>KCND3, elav>KCND3-∆F227 and elav>KCND3-G345V flies were all shorter than elav-gal4 control flies at room temperature condition (Fig. 2C). Through climbing analysis we found that the motor functions of elav>KCND3, elav>KCND3-∆F227 and elav>KCND3-G345V flies were reduced when compared with elav-gal4 control flies. 12.

(14) (Fig. 2D). Additionally, pan-neuronal overexpression of either wild type or mutant KCND3 mutants reduced the lifespan and motor function of flies. For instance, overexpression of either wild type or mutant KCND3 under the control of elav-GAL4 caused mild retinal degeneration and pigment loss phenotypes at room temperature (Fig. 3). The depigmentation phenotype in KCND3-∆F227 and KCND3-G345V expressing flies seem more severe than that of normal KCND3 expressing flies in early age of one-day after eclosion. The Elav-gal4 control flies did not show any depigmentation phenotype even at old age of 60-day after eclosion. Overexpression of either wild type or mutant KCND3 (KCND3-∆F227, KCND3-G345V) in Drosophila eyes driven by GMR-GAL4 driver did not induce photoreceptor degeneration at 25oC in the early age (Fig. 4). The retinal structures of all transgenic flies and GMR-gal4 transgenic flies were all normal when raised at 25oC science one-day till 30-day after eclosion. At the old age 45-day after eclosion start to show mild degeneration of depigmentation. It well perform the progressive neurodegeneration feature as SCA pathology. Enhance the transgene expression In the beginning of our experiment lake of degeneration in the retinal cell. We suspected that the reason that the lack of retinal degeneration phenotype of ectopic KCND3 expression by GMR-GAL4 could be due to the insufficient expression of transgenes. To test this, we increased the expression level of transgene by raised the transgenic flies at 29oC simultaneously. We found that overexpression of either wild type or mutant KCND3-∆F227 and KCND3-G345V driven by GMR-Gal4 induced severe eye degeneration in newly eclosed flies at 29oC (Fig. 5). Neurodegeneration in Drosophila is commonly accompanied by the formation of vacuoles in brain. To better correlate the extent of neuronal apoptosis, we examined the brain structure of our transgenic flies using histological approaches. In Drosophila 13.

(15) brain, the basophilic cell bodies of neurons and glia which occupy the outer cortex are stained dark purple by H&E while the eosinophilic process that located in the inner neuropil region are pink or light purple after staining. We found no anatomic abnormality in the nervous system of young control elav-GAL4 flies (Fig. 6A). In contrast to control flies, ectopic overexpression KCND3 transgenic flies showed obviously vacuolization in both cortex and neuropil (Fig. 6B-D). Besides, the number and size of vacuoles increased with age, suggest that the neurodegeneration is progressive (Fig. 6E-H). Acridine orange is a vital dye that specifically cells undergoing apoptosis in Drosophila and has been shown to be specific for apoptotic cells rather than label the chromatin of cells dying by oxygen starvation or necrosis. To analyze the phenotype of neurodegeneration in Drosophila effectively, the brain structure was revealed then process acridine orange stain and observed using confocal microscopy. We determined that enhance of the transgene did cause obvious damage in the brain cells of flies expressing KCND3. (Fig. 7) The range of death cells in the brain of the transgene flies were more numerous and also can see foci formation. Together with the observation above, shows that the disease fly model recapitulates many characteristic feature of SCA22, and is therefore a suitable model to study pathogenesis of the neuronal disorder. Ectopic expression of KCND3 mutant gene will cause Apoptosis which lead to neurodegeneration Previous studies revealed that KCND3-ΔF227 mutant protein did not move to the plasma membrane and retained in the endoplasmic reticulum (ER) [16]. We suspect this may lead to ER stress that have been identified as a cause of cell death. Besides, KCND3 encode potassium channel that will interrupt the balance between Na+/K+ if ectopic express that proposed to be a key early stage in induction of apoptosis. To 14.

(16) further demonstrate the ectopic KCND3 phenotype is amenable to progress cell death, we determined the retinal phenotype of control and KCND3 transgenic flies cross with Drosophila inhibitor of apoptosis GAL4-driver GMR>DIAP2 line to directly test. We found that the retinal structure did rescue at 29oC (Fig. 8). Thus our finding suggesting that ectopic KCND3 neurodegeneration is caused by apoptosis.. 15.

(17) Discussion. To address the origin of the locomotion defects in KCND3 mutants, the UAS/GAL4 system was used to overexpress wild type or mutant KCND3 in defined tissues. Two GAL4 lines GMR-GAL4 and elav-GAL4 that drive KCND3 overexpression were found to reduce the survivorship, mobility of mutants and brain cell loss suggesting that ectopic KCND3 expression may lead to neurodegeneration. We recapitulates many characteristic feature of SCA22, such as age-dependent neurodegeneration, including retain degeneration, brain vacuolization, mobility defect, and shorten lifespan, therefore could be a suitable model to study pathogenesis of the neuronal disorder. Moreover, KCND3-ΔF227 shows different degeneration manner driver between GMR-GAL4 and evav-GAL4. The ectopic expression phenotype of KCND3-ΔF227 work much obvious in neuronal elav-GAL4 driver. Contrary, the ectopic expression phenotype of KCND3-G345V show neurodegeneration in both elav-GAL4 and GMR-GAL4 driver. Suggest the two KCND3 mutant may have different pathogenic mechanism. We hypnotized that KCND3 expression may cause loss of neurons through apoptosis. To further proof our hypothesis, we raise transgenic flies at elevated temperature at 29oC ensure that the ectopic expression well. As mentioned above, KCND3 is a potassium channel, and the mutant Kv4.3-ΔF227 is co-localized with ER, we suspect that the pathogenesis of SCA22 is likely to be mediated through ER stress or K+ efflux induced apoptosis.Although this study did not test directly the pathogenic mechanism of ER stress or K+ efflux. We direct show ectopic expression both KCND3-ΔF227 and KCND3-G345V will lead to apoptosis.. 16.

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(23) Figure 1. Transgenic constructs of human KCND3/Kv4.3. The expression constructs were cloned into the EcoRI and NotI sites of the pBID-UASC plasmid. The expression of the transgenes was under the control of UAS promoter (10X UAS). The vector also contains a phiC31 attB sequence which would allow site-specific recombination. The transgene constructs were introduced into the fly genomes through germ-line transformation procedure.. A. B 22.

(24) C. D. 100. 5. Perscent survial. 80 Perscent survial. 6. Elav-gal4 KCND3G345V KCND3DF227 KCND3. 60 40. **. *** *. *** *. 4. ** *. *** *. **. 3 2 1. 20. 0. 0. *** **. KCND3DF227 KCND3. Elav-gal4 KCND3G345V. 1. 10 20 30 40 50 60 70 80 Age (days after eclosion). 1. 15 30 Age (days after eclosion). Figure 2. The KCND3 transgenic flies model. (A) (B)Western blotting analysis demonstrated that the expression of KCND3-ΔF227 mutant protein was significantly less that of wild type or G345V variants. β-Actin served as the internal loading control. (C) Lifespans were determined on standard cornmeal-yeast-agar medium at room temperature. Neuronal overexpression of wild type or mutant KCND3 in Drosophila reduced lifespan and mobility. (D) Mutant KCND3 expression also reduced the climbing ability of Drosophila at early adulthood.. 23.

(25) elav-GAL4/+. elav>. elav>. elav>. KCND3 WT. KCND3-G345V. KCND3-∆F227. Day 1. Day 15. Day 60. Figure 3. Neuronal overexpression of either wild type or mutant KCND3 induced mild retinal depigmentation at 25oC. Overexpressing KCND3-∆F227 and KCND3-G345V in neuronal system of Drosophila driven by elav-GAL4 at 25oC lead to retinal depigmentation in one-day old flies.. 24.

(26) GMR-GAL4/+. GMR> KCND3 WT. GMRr>. GMR>. KCND3-G345V. KCND3-∆F227. Day 1. Day 15. Day 30. Day 45. Figure 4. Retinal overexpression of wild type and mutant KCND3 in Drosophila at 25oC. Overexpressing KCND3-∆F227 and KCND3-G345V in eye of Drosophila driven by GMR-GAL4 at room temperature did not show retinal degeneration in the early age fly eye. Mild degeneration of depigmentation was observed in the old age of 45-day elav-GAL4 driven transgene flies.. 25.

(27) GMR-GAL4/+. GMR> KCND3 WT. GMRr>. GMR>. KCND3-G345V. KCND3-∆F227. Day 1. Day 15. Day 30. Day 45. Figure 5. Retinal overexpression of wild type or mutant induces photoreceptor degeneration at 29oC. Overexpressing KCND3-∆F227 and KCND3-G345V in eye of Drosophila by GMR-GAL4 at 29oC caused retinal degeneration at newly eclosed flies.. 26.

(28) Days after eclosion 1. 15. E. B. F. C. G. D. H. elav> KCND3-∆F227. elav> KCND3-G345V. elav>KCND3 WT. elav-GAL4/+. A. Figure 6. Ectopic KCND3 caused neuropathies in fly brain at 29oC. Age-dependent neurodegeneration was revealed by H&E staining of frontal brain section. (A) Neurodegeneration was not detected in one-day old control elav-GAL4 flies. (B-D) Mild vacuolar degeneration (arrow) was observed in one-day old of 27.

(29) KCND3 transgene flies. (E) Mild vacuolar degeneration was observed in aged control elav –GAL4 flies. (F-H) Larger and more vacuoles were present in the cortex of aged KCND3 transgenic flies.. 28.

(30) Figure 7. Overexpression of KCND3 or KCND3 mutant induced apoptosis in the brains at 29oC. Confocal images of Acridine orange staining of brains from the flies at newly eclosed age. Neurodegeneration was not detected in control elav-GAL4 flies. Serious degeneration of brain cell death vacuolar was observed in KCND3 transgene flies.. 29.

(31) Gmr-gal4. KCNDWT. KCNDG345V. KCNDDF227. DIAP2. DIAP2+KCNDWT. DIAP2+KCNDG345V. DIAP2+KCNDDF227. Figure 8. The Drosophila inhibitor of apoptosis (IAP) rescue neuropathies on retinal cells at 29oC. The eye phenotype caused by ectopic KCND3 is rescued by expression of GMR>DIAP2 driver.. 30.

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