Biology subjects

Kim, N. C.

Publications and source records attributed to Kim, N. C..

7 recordsLinked to original sources

bioRxiv2025

JAG2-related muscular dystrophy and Notch signaling dysfunction in muscle stem cells

We previously identified a muscular dystrophy caused by biallelic variants in JAG2, whose protein product Jagged2 is a canonical Notch ligand. However, the disease mechanism remains unclear, particularly with respect to muscle stem cell (MuSC) function and muscle regeneration. We examined the consequences of JAG2 deficiency and modeled pathogenic JAG2 variants in vitro and in vivo, the latter in mouse and fly models and with particular attention to the MuSC-muscle endothelial cell (MuEC) niche. We found that both Jag2 deficiency and overexpression of pathogenic JAG2 variants impaired Notch signaling and myogenic self-renewal and differentiation. Hypomorphic Jag2 mutant (Jag2sm) mice display depleted MuSCs, corresponding with impaired muscle regeneration in those mice. Co-culture experiments and the examination of cell-type-specific Jag2 conditional knockout mice demonstrated that MuEC-specific Jag2 knockout resulted in reduced MuSC self-renewal, while MuSC-specific Jag2 knockout resulted in reduced myogenic differentiation. Human reference JAG2, but not human pathogenic variants of JAG2, rescued the deficiency of Serrate (Ser), the Drosophila ortholog of JAG2. Therefore, pathogenic variants in JAG2 impair muscle development and regeneration through disrupted cell-autonomous cis- inhibition and non-autonomous trans-activation involving Notch signaling dysfunction. Our findings indicate that optimizing JAG2-mediated Notch signaling is a potential therapeutic approach for JAG2-related muscular dystrophy.

developmental biology↗

bioRxiv2024

FDA approved PDE4 inhibitors reduce the dominant toxicity of ALS/FTD associated CHCHD10S59L

Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10(CHCHD10) have been identified as a genetic cause of amyotrophic lateral sclerosis and/or frontotemporal dementia(ALS-FTD). In our previous studies using in vivo Drosophila model expressing C2C10HS81L, and human cell models expressing CHCHD10S59L, we have identified that the PINK1/Parkin pathway is activated and causes cellular toxicity. Furthermore, we demonstrated that pseudo-substrate inhibitors for PINK1 and mitofusin2 agonists mitigated the cellular toxicity of CHCHD10S59L. Evidences using in vitro/ in vivo genetic and chemical tools indicate that inhibiting PINK1 would be the most promising treatment for CHCHD10S59L-induced diseases. Therefore, we have investigated cellular pathways that can modulate the PINK1/Parkin pathway and reduce CHCHD10S59L-induced cytotoxicity. Here, we report that FDA-approved PDE4 inhibitors reduced CHCHD10S59L-induced morphological and functional mitochondrial defects in human cells and an in vivo Drosophila model expressing C2C10HS81L. Multiple PDE4 inhibitors decreased PINK1 accumulation and downstream mitophagy induced by CHCHD10S59L. These findings suggest that PDE4 inhibitors currently available in the market may be repositioned to treat CHCHD10S59L-induced ALS-FTD and possibly other related diseases.

neuroscience↗

bioRxiv2024

Orange Maker: a CRISPR/Cas9-mediated genome editing and screening project to generate orange-eyed DarkJedi GAL4 lines by undergraduate students

One of the greatest strengths of Drosophila genetics is its easily observable and selectable phenotypic markers. The mini-white marker has been widely used as a transgenic marker for Drosophila transgenesis. Flies carrying a mini-white construct can exhibit various eye colors ranging from pale orange to intense red, depending on the insertion site and gene dosage. Because the two copies of the mini-white marker show a stronger orange color, this is often used for selecting progenies carrying two transgenes together in a single chromosome after chromosomal recombination. However, some GAL4 lines available in the fly community originally have very strong red eyes. Without employing another marker, such as GFP, generating a recombinant chromosome with the strong red-eyed GAL4 and a desired UAS-transgene construct may be difficult. Therefore, we decided to change the red eyes of GAL4 lines to orange color. To change the eye color of the fly, we tested the CRISPR/Cas9 method with a guide RNA targeting the white gene with OK371-GAL4 and elav-GAL4. After a simple screening, we have successfully obtained multiple lines of orange-eyed OK371-GAL4 and elav-GAL4 that still maintain their original expression patterns. All of these simple experiments were performed by undergraduate students, allowing them to learn about a variety of different genetic experiments and genome editing while contributing to the fly research community by creating fruit fly lines that will be used in real-world research.

genetics↗

bioRxiv2023

The 21-base pair deletion mutant Calpain 3 does not inhibit wild-type Calpain 3 activity

IntroductionCalpain 3 is one of the calpain protease family members, which is a calcium-dependent proteolytic enzyme predominantly expressed in skeletal muscle. Loss-of-function mutations in the Calpain 3 gene have been related to autosomal recessive Limb-Girdle Muscular Dystrophy 1 (LGMDR1), a common form of muscular dystrophy. Recently, the heterozygous 21-bp deletion mutation of the Calpain 3 gene has been reported to cause autosomal dominant Limb-Girdle Muscular Dystrophy 4 (LGMDD4). According to its dominant inheritance pattern, it has been suggested that the deletion mutant proteins act in a dominant-negative manner. Therefore, we examined whether the mutant protein can suppress the activity of wild-type Calpain 3 and has any dominant toxicity in cell culture and in vivo Drosophila models. MethodsA human cell culture (HeLa cells) model with the transient transfection of human wild-type and mutant Calpain 3 and in vivo Drosophila models overexpressing wild-type and mutant Drosophila Calpain A and B were utilized in this study to assess dominant effects of Calpain 3 21-bp deletion mutant. Western blot analysis was used to determine protein stability and catalytic activity in cell culture. External eye morphology and muscle integrity were examined to observe dominant toxicity in Drosophila models. ResultsThe 21-bp deletion mutation of Calpain 3 resulted in catalytic inactivation, which did not inhibit wild-type Calpain 3 autolytic and catalytic activity against Calpastatin in HeLa cells. In addition, the mutant protein was normally processed by wild-type Calpain 3. Overexpression of wild-type and deletion mutant Calpain 3 in the Drosophila eye and muscles did not exhibit significant developmental and age-related dominant toxicity. DiscussionWe provide evidence that mutant Calpain 3 does not suppress wild-type Calpain 3 activity. Rather, it is a mutant lacking autocatalytic processing activity like many other loss-of-function Calpain 3 mutants causing LGMDR1. Our results implicate that the stability of the heteromeric mutant and wild-type Calpain 3 complexes may be affected without inhibiting the wild-type activity per se. However, a more thorough investigation is necessary to understand the molecular mechanism and dominant inheritance of the heterozygous 21-bp deletion mutation in LGMDD4.

cell biology↗

bioRxiv2020

Eip74EF is a dominant modifier for ALS-FTD linked mutant VCP phenotypes in Drosophila, but not miR-34

In 2012 Liu et al. reported that miR-34 is an age-related miRNA regulating age-associated events and long-term brain integrity in Drosophila. They demonstrated that modulating miR-34 and its downstream target Eip74EF showed beneficial effects on age-related diseases using a Drosophila model of SCA3trQ78. These results imply that miR-34 could be a general genetic modifier and therapeutic candidate for age-related diseases. Therefore, we examined the effect of miR-34 and Eip74EF on another age-related Drosophila disease model. Using a Drosophila model expressing mutant Drosophila VCP that causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), or multisystem proteinopathy (MSP), we demonstrated that abnormal eye phenotypes generated by Drosophila VCP R152H were rescued when expressed with Eip74EF siRNA. Contrary to our expectation, miR-34 overexpression resulted in lethality when expressed with mutant VCP. Our data indicate that the other downstream targets of miR-34 might more significantly interact with mutant VCP, causing lethality. Identifying transcriptional targets of Eip74EF might provide valuable insights into diseases caused by mutations in VCP such as ALS, FTD, and MSP.

pathology↗

bioRxiv2019

Dominant toxicity of ALS-FTD-associated CHCHD10S59L is mediated by TDP-43 and PINK1

Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) are a genetic cause of amyotrophic lateral sclerosis and/or frontotemporal dementia (ALS-FTD). To elucidate how mutations in CHCHD10 induce disease, we generated a Drosophila melanogaster model of CHCHD10-mediated ALS-FTD. Expression of CHCHD10S59L in Drosophila caused gain-of-function toxicity in eyes, motor neurons, and muscles, in addition to mitochondrial defects in flies and HeLa cells. TDP-43 and PINK1 formed two axes, driving the mutant-dependent phenotypes. CHCHD10S59L expression increased TDP-43 insolubility and mitochondrial translocation. Blocking mitochondrial translocation with a peptide inhibitor reduced CHCHD10S59L-mediated toxicity. PINK1 knockdown rescued CHCHD10S59L-mediated phenotypes in Drosophila and HeLa cells. The two PINK1 substrates mitofusin and mitofilin were genetic modifiers of this phenotype. Mitofusin agonists reversed the CHCHD10S59L-induced phenotypes in Drosophila and HeLa cells and increased ATP production in Drosophila expressing C9orf72 with expanded GGGGCC repeats. Two peptides inhibitors of PINK1 mitigated the mitochondrial defects introduced by CHCHD10S59L expression. These findings indicate that TDP-43 mitochondrial translocation and chronic activation of PINK1-mediated pathways by CHCHD10S59L generate dominant toxicity. Therefore, inhibiting PINK1 activity may provide a therapeutic strategy for CHCHD10-associated disease.\n\nOne Sentence SummaryInhibition of TDP-43 mitochondrial translocation or PINK1 kinase activity mitigates CHCHD10S59L-mediated mitochondrial toxicity.

neuroscience↗

bioRxiv2016

Metabolome Identification by Systematic Stable Isotope Labeling Experiments and False Discovery Analysis with a Target-Decoy Strategy

We introduce a formula-based strategy and algorithm (JUMPm) for global metabolite identification and false discovery analysis in untargeted mass spectrometry-based metabolomics. JUMPm determines the chemical formulas of metabolites from unlabeled and stable-isotope labeled metabolome data, and derives the most likely metabolite identity by searching structure databases. JUMPm also estimates the false discovery rate (FDR) with a target-decoy strategy based on the octet rule of chemistry. With systematic stable isotope labeling of yeast, we identified 2,085 chemical formulas (10% FDR), 892 of which were assigned with metabolite structures. We evaluated JUMPm with a library of synthetic standards, and found that 96% of the formulas were correctly identified. We extended the method to mammalian cells with direct isotope labeling and by heavy yeast spike-in. This strategy and algorithm provide a powerful a practical solution for global identification of metabolites with a critical measure of confidence.

biochemistry↗