Biology subjects

Kanca, O.

Publications and source records attributed to Kanca, O..

15 recordsLinked to original sources

bioRxiv2026

CDKL5 phosphorylates neuronal ELAVL proteins to promote mRNA binding, protein synthesis and visual cortex development

Loss-of-function mutations in the X-linked CDKL5 gene lead to a severe neurodevelopmental disorder characterized by early-onset epilepsy, known as CDKL5 Deficiency Disorder (CDD). Despite its clinical significance, the physiological substrates of the serine/threonine kinase CDKL5 and its roles in neuronal development remain poorly understood. To address this, we performed quantitative phosphoproteomics analysis in Cdkl5 knockout (KO) mouse brains, identifying 22 CDKL5 substrates involved in diverse cellular functions. Among these, we focused on the neuronal RNA-binding proteins (nELAVLs) ELAVL2, ELAVL3, and ELAVL4, as these represented the only evolutionarily conserved phosphorylation and are known regulators of neuronal differentiation. Through kinase assays and individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP), we found that CDKL5 phosphorylates S119/131 in ELAVL2/3/4, promoting their cytoplasmic localization and enhancing their binding to target mRNAs at 3UTRs. Loss of CDKL5 activity in neurons caused reduced new protein synthesis, as measured by puromycin incorporation; this phenotype was rescued by knockdown of the nELAVL inhibitor long non-coding RNA, RNY3, revealing an essential function of CDKL5 in enhancing protein synthesis via nELAVL phosphorylation. To investigate the in vivo functions of nELAVL phosphorylations, we generated Elavl2/3/4 phosphomutant mice and found that collectively nELAVL phosphorylations are required for viability. Proteomic and transcriptomic analyses of Elavl2/3 homozygous phosphomutants, which exhibited sub-viability, revealed compensatory upregulation of ELAVL4 and synaptic proteins. Functionally, in Elavl2/3/4 triple heterozygous mice Neuropixels recordings in the primary visual cortex showed deficits in receptive field properties and orientation tuning, revealing the role of nELAVL phosphorylation for accurate cortical circuit formation. Our study uncovers a crucial role for CDKL5 in regulating nELAVL-mediated protein synthesis and the development of cortical circuits.

neuroscience↗

bioRxiv2026

NLGN3 autism variants have distinct functional impact on synapses and sleep behavior in Drosophila

Neuroligin-3 (NLGN3) was first identified as a risk gene associated with autism spectrum disorder (ASD). The initial variant, p.R451C, associating NLGN3 with ASD has been heavily investigated, yet little is known about the functional consequences of other NLGN3 variants. Furthermore, while most of the identified variants are present in males with maternally inherited variants from unaffected mothers, several de novo variants were observed in females, suggesting a possible functional difference between de novo and maternally inherited variants. To address the functional consequences of NLGN3 variants in vivo, we generated transgenic Drosophila models corresponding to one de novo variant (p.R175W) identified in one female proband, and two maternally inherited variants (p.R451C and p.R597W) identified in male probands. In Drosophila, loss of the fly homolog, Nlg3, altered sleep patterns, synaptic architecture, and vesicle dynamics, which were rescued by the expression of the human NLGN3Ref allele. When comparing the variants, the de novo p.R175W variant and the maternally inherited p.R451C variant altered synapse morphology and sleep patterns, with minimal effects on vesicle dynamics, and the p.R597W variant altered sleep and vesicle dynamics with minimal impact on synapse morphology. Using overexpression models, human NLGN3Ref altered sleep patterns and synaptic morphology. Moreover, the p.R175W variant exacerbated sleep phenotypes, and the p.R175W and p.R451C variants exacerbated synapse morphology phenotypes. Together, our findings suggest that de novo NLGN3 variants identified in females are likely gain-of-function, while maternally inherited variants have mixed loss-and gain-of-function effects. Moreover, the location of the variants may contribute to the distinct functional differences we observed. Some NLGN3 variants disrupt synaptic development, while other variants alter synaptic function, suggesting that NLGN3 variants have differential effects. These functional differences may provide insight into the heterogeneity of individuals with ASD. Author SummaryAutism spectrum disorder (ASD) is a common neurodevelopmental disorder. Mutations in the Neuroligin-3 (NLGN3) gene are associated with ASD but very few of these mutations have been characterized in animal models. Most of these mutations affect male individuals who maternally inherited their genetic mutation; however, more rarely female individuals may present with a genetic mutation that was not identified in either of the parents. Here, we utilized the fruit fly model to investigate how three different mutations, one mutation identified in a female and two mutations identified in males, affect the flys behavior and synapse development. We identified altered sleep patterns in some of our mutants which is consistent with sleep disturbances being highly comorbid with ASD. Additionally, we identified alterations in synapse development and function which is consistent with the role of NLGN3 in synapse formation and maturation. Together, our findings support that NLGN3 is important for regulating the synapse and mutations in this gene can alter its function. However, different mutations can have differential effects. This demonstrates the need to assess multiple variants simultaneously because each variant may have distinct functional significances.

genetics↗

bioRxiv2025

Revealing the nervous system requirements of Alzheimer disease risk genes in Drosophila

Most Alzheimers disease (AD) susceptibility genes have poorly understood roles in the central nervous system (CNS). To address this gap, we systematically characterized 100 conserved candidate AD risk genes using a cross-species strategy in the fruit fly, Drosophila melanogaster. Genes were prioritized based primarily on human functional genomic evidence. We generated custom, loss-of-function alleles for each of the conserved fly orthologs. Most of the genes (80%) are expressed in the adult brain, including 24 neuron- and 13 glia-specific expression patterns. Overall, we identify 50 candidate AD risk gene homologs with requirements for CNS structure or function, including 18 whose loss of function causes neurodegeneration (e.g., Snx6/SNX32 and ClC-a/CLCN1), 35 required for neurophysiology (e.g., Arr1/ARRB2, stai/STMN4), and 8 with diminished CNS resilience following a thermal or mechanical stress (e.g., cindr/CD2AP, Amph/BIN1). In a parallel screen, we found 28 AD risk gene homologs (e.g, Ets98B/SPI1, Yod1/YOD1) that modify the neurotoxicity of either amyloid-{beta} peptide or tau protein, which aggregate to form AD pathology. To translate our findings back to human AD, we developed and deployed oligogenic risk scores based on gene clusters with shared nervous system phenotypes in flies, pinpointing functional pathways that differentially drive AD risk. Our results--available online via the Alzheimers Locus Integrative Cross-species Explorer (alice.nrihub.org)--reveal novel nervous system requirements for dozens of AD risk genes and may enable dissection of causal heterogeneity in AD.

genetics↗

bioRxiv2024

Distinguishing PEX gene variant severity for mild, severe, and atypical peroxisome biogenesis disorders in Drosophila

Peroxisomal biogenesis disorders (PBD) are autosomal recessive disorders caused by loss-of-function mutations of one of the PEX genes responsible for peroxisomal formation. Impaired peroxisome assembly causes severe multisystemic failure with patient phenotypes ranging from epilepsy, liver disease, feeding issues, biochemical abnormalities, and neurodegeneration. Variants in the same PEX gene can produce wide differences in severity, ranging from individuals with death in the first year of life to adults with milder complications. To study this strong genotype-phenotype correlation, we selected specific human PEX gene mutations and utilized Drosophila as a model organism. We generated flies replacing the coding sequence of our Pex gene of interest with a KozakGAL4 (KZ) promoter trap sequence. These cassettes simultaneously knock-out of the Pex gene and knock-in a GAL4 driver, ideal for making "humanized" flies in which the human PEX gene can replace the fly loss. We assessed Pex2KZ and Pex16KZ lines in lifespan, bang sensitivity, and climbing assays and confirmed that these are strong loss-of-function alleles. In parallel, we generated human reference and variant UAS-cDNA lines of PEX2 and PEX16 variants in Drosophila. We observed nearly complete phenotypic rescue of Drosophila Pex2 and Pex16 loss when human PEX2Refor PEX16Ref, respectively, were expressed. We also provide evidence for an allele severity spectrum in PEX2 and PEX16 in which some missense alleles, such as PEX2C247R, are equally severe as early truncations, such as PEX2R119*. We also observed that alleles associated with mild PBD, such as PEX2E55K, show variability depending on the assay but do not fully rescue. Finally, alleles associated with atypical ataxia phenotypes, such as PEX16F332Del, can perform as well as PEX16Ref, depending on the assay. Altogether, these Drosophila lines effectively model the range of severity of peroxisomal biogenesis disorders.

genetics↗

bioRxiv2024

Flow zoometry of Drosophila

Drosophila serves as a highly valuable model organism across numerous fields including genetics, immunology, neuroscience, cancer biology, and developmental biology. Central to Drosophila-based biological research is the ability to perform comprehensive genetic or chemical screens. However, this research is often limited by its dependence on laborious manual handling and analysis, making it prone to human error and difficult to discern statistically significant or rare events amid the noise of individual variations resulting from genetic and environmental factors. In this article we present flow zoometry, a whole-animal equivalent of flow cytometry for large-scale, individual-level, high-content screening of Drosophila. Our flow zoometer automatically clears the tissues of Drosophila melanogaster, captures three-dimensional (3D) multi-color fluorescence tomograms of single flies with single-cell volumetric resolution at an unprecedented throughput of over 1,000 animals within 48 hours (24 hr for clearing; 24 hr for imaging), and performs AI-enhanced data-driven analysis - a task that would traditionally take months or years with manual techniques. To demonstrate its broad applications, we employed the flow zoometer in various laborious screening assays, including those in toxicology, genotyping, and tumor screening. Flow zoometry represents a pivotal evolution in high-throughput screening technology: previously from molecules to cells, now from cells to whole animals. This advancement serves as a foundational platform for "statistical spatial biology", to improve empirical precision and enable serendipitous discoveries across various fields of biology.

bioengineering↗

bioRxiv2024

Loss of function in RBBP5 results in a syndromic neurodevelopmental disorder associated with microcephaly

PurposeEpigenetic dysregulation has been associated with many inherited disorders. RBBP5 encodes a core member of the protein complex that methylates histone 3 lysine-4 (H3K4) and has not been implicated in human disease. MethodsWe identify five unrelated individuals with de novo heterozygous pathogenic variants in RBBP5. Three truncating and two missense variants were identified in probands with neurodevelopmental symptoms including global developmental delay, intellectual disability, microcephaly, and short stature. Here, we investigate the pathogenicity of the variants through protein structural analysis and transgenic Drosophila models. ResultsBoth missense p.T232I and p.E296D variants affect evolutionarily conserved amino acids and are expected to interfere with the interface between RBBP5 and the histones. In Drosophila, ubiquitous overexpression of human RBBP5 is lethal in the larval developmental stage. Loss of Rbbp5 leads to a reduction in brain size, and the human reference, p.T232I, or p.E296D variant transgenes fail to rescue loss of Rbbp5. Expression of either missense variant in an Rbbp5 null background results in a less severe microcephaly phenotype than the human reference, indicating both p.T232I and p.E296D variants are loss-of-function alleles. ConclusionDe novo heterozygous variants in RBBP5 are associated with a syndromic neurodevelopmental disorder. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/578086v1_ufig1.gif" ALT="Figure 1"> O_LINKSMALLFIG WIDTH=200 HEIGHT=24 SRC="FIGDIR/small/578086v1_ufig1a.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@11337daorg.highwire.dtl.DTLVardef@b7f4d8org.highwire.dtl.DTLVardef@12bc283org.highwire.dtl.DTLVardef@1ef4e09_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2023

Loss of Mitochondrial Enoyl CoA Reductase causes elevated ceramide levels and impairs iron metabolism

In most eukaryotic cells fatty acid synthesis occurs in the cytoplasm as well as in mitochondria. However, the relative contribution of mitochondrial fatty acid synthesis (mtFAS) to the cellular lipidome of metazoans is ill-defined. Hence, we studied the function of the fly Mitochondria enoyl CoA reductase (Mecr), the enzyme required for the last step of mtFAS. Loss of mecr causes lethality while neuronal loss leads to progressive neurological defects. We observe an elevated level of ceramides, a defect in Fe-S cluster biogenesis and increased iron levels in mecr mutants. Reducing the levels of either iron or ceramide suppresses the neurodegenerative phenotypes indicating that increased ceramides and iron metabolism are interrelated and play an important role in the pathogenesis. Mutations in human MECR cause pediatric-onset neurodegeneration and patient-derived fibroblasts display similar elevated ceramide levels and impaired iron homeostasis. In summary, this study shows an as-yet-unidentified role of mecr/MECR in ceramide and iron metabolism providing a mechanistic link between mtFAS and neurodegeneration.

neuroscience↗

bioRxiv2022

De Novo Variants in MRTFB have gain of function activity in Drosophila and are associated with a novel neurodevelopmental phenotype with dysmorphic features.

Myocardin-Related Transcription Factor B (MRTFB) is an important transcriptional regulator which promotes the activity of an estimated 300 genes during different stages of development. Here we report two pediatric probands with de novo variants in MRTFB (R104G and A91P) and mild dysmorphic features, intellectual disability, global developmental delays, speech apraxia, and impulse control issues. As the MRTFB protein is highly conserved between vertebrate and invertebrate model organisms, we generated a humanized Drosophila model expressing the human MRTFB protein in the same spatial and temporal pattern as the fly gene. Expression of the human MRTFBR104G variant using a mrtf-T2A-GAL4 line proved to be embryonic lethal. Additional phenotypes were also identified by expressing the MRTFBR104G and MRTFBA91P variant in a subset of Drosophila tissues. Notably, expression within wing tissues resulted in an expansion of intervein tissue, wing vein thickening, shortening or loss of wing veins, and blistering. The MRTFBR104G and MRTFBA91P variants also display a decreased level of actin binding within critical RPEL domains, resulting in increased transcriptional activity and changes in the organization of the Actin cytoskeleton. These changes were not observed in flies expressing two additional candidate variants, MRTFBN95Sand MRTFBR109Q, highlighting that the location of the mutation within the 2nd RPEL domain is critical to the pathogenicity of the variant. These changes suggest that the MRTFBR104G and MRTFBA91P alleles we have identified affect the regulation of the protein and that these variants in MRTFB underly a novel neurodevelopmental disorder.

genetics↗

bioRxiv2021

An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR mediated homologous recombination

Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination (Lee et al., 2018). These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100-200 bps) (Kanca et al., 2019a). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 50% of Drosophila genes lack suitable coding introns for integration of artificial exons. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon-intron structure, with a 70-80% success rate.

genetics↗

bioRxiv2021

Systematic expression profiling of dprs and DIPs reveals cell surface codes in Drosophila larval peripheral neurons

In complex nervous systems, neurons must identify their correct partners to form synaptic connections. The prevailing model to ensure correct recognition posits that cell surface proteins (CSPs) in individual neurons act as identification tags. Thus, knowing what cells express which CSPs would provide insights into neural development, synaptic connectivity, and nervous system evolution. Here, we investigated expression of dprs and DIPs, two CSP subfamilies belonging to the immunoglobulin superfamily (IgSF), in Drosophila larval motor neurons (MNs), sensory neurons (SNs), peripheral glia and muscles using a collection of GAL4 driver lines. We found that dprs are more broadly expressed than DIPs in MNs and SNs, and each examined neuron expresses a unique combination of dprs and DIPs. Interestingly, many dprs and DIPs are not robustly expressed, but instead, are found in gradient and temporal expression patterns. Hierarchical clustering showed a similar expression pattern of dprs and DIPs in neurons from the same type and with shared synaptic partners, suggesting these CSPs may facilitate synaptic wiring. In addition, the unique expression patterns of dprs and DIPs revealed three uncharacterized MNs - MN23-Ib, MN6-Ib (A2) and MN7-Ib (A2). This study sets the stage for exploring the functions of dprs and DIPs in Drosophila MNs and SNs and provides genetic access to subsets of neurons.

neuroscience↗

bioRxiv2021

In vivo regulation of fluorescent fusion proteins by engineered kinases

Reversible protein phosphorylation by kinases in extensively used to control a plethora of processes essential for proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase-substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase-substrate by pleiotropic effects. To overcome this limitation, we have created protein binder-based engineered kinases for direct, robust and tissue-specific phosphorylation of target fluorescent protein fusions in vivo. We show that synthetic Rok kinases, based on the Drosophila ortholog of Rho-associated protein kinase (ROCK), are functional enzymes and can activate myosin II through phosphorylation of Sqh::GFP or Sqh::mCherry in different morphogenetic processes in a developing fly embryo. We next use the system to study the impact of actomyosin activation specifically in the developing tracheal branches and showed that ectopic activation of actomyosin with engineered Rok kinase did not prevent cell intercalation nor the formation of autocellular junctions. We assume that this approach can be adapted to other kinases and targets in various eukaryotic genetic systems.

developmental biology↗

bioRxiv2021

Drosophila functional screening of de novo variants in autism uncovers deleterious variants and facilitates discovery of rare neurodevelopmental diseases

Individuals with autism spectrum disorders (ASD) exhibit an increased burden of de novo variants in a broadening range of genes. We functionally tested the effects of ASD missense variants using Drosophila through humanization rescue and overexpression-based strategies. We studied 79 ASD variants in 74 genes identified in the Simons Simplex Collection and found 38% of them caused functional alterations. Moreover, we identified GLRA2 as the cause of a spectrum of neurodevelopmental phenotypes beyond ASD in eight previously undiagnosed subjects. Functional characterization of variants in ASD candidate genes point to conserved neurobiological mechanisms and facilitates gene discovery for rare neurodevelopmental diseases.

genetics↗

bioRxiv2019

An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms

We previously reported a CRISPR-mediated knock-in strategy into introns of Drosophila genes, generating an attP-FRT-SA-T2A-GAL4-polyA-3XP3-EGFP-FRT-attP transgenic library for multiple uses (Lee et al., 2018b). The method relied on double stranded DNA (dsDNA) homology donors with [~]1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely in vivo. The approach is fast, cheap, and scalable.

genomics↗

bioRxiv2018

An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila

We generated new genetic tools to efficiently tag genes in Drosophila. Double Header (DH) utilizes intronic MiMIC/CRIMIC insertions to generate artificial exons for GFP mediated protein trapping or T2A-GAL4 gene trapping in vivo based on CRE recombinase to avoid embryo injections. DH significantly increases integration efficiency compared to previous strategies and faithfully reports the expression pattern of genes and proteins. The second technique targets genes lacking coding introns using a two-step cassette exchange. First, we replace the endogenous gene with an excisable compact dominant marker using CRISPR making a null allele. Second, the insertion is replaced with a protein::tag cassette.\n\nThis sequential manipulation allows the generation of numerous tagged alleles or insertion of other DNA fragments that facilitates multiple downstream applications. Both techniques allow precise gene manipulation and facilitate detection of gene expression, protein localization and assessment of protein function, as well as numerous other applications.

genetics↗

bioRxiv2018

Loss-of-function in IRF2BPL is associated with neurological phenotypes

The Interferon Regulatory Factor 2 Binding Protein Like (IRF2BPL) gene encodes a member of the IRF2BP family of transcriptional regulators. Currently the biological function of this gene is obscure, and the gene has not been associated with a Mendelian disease. Here we describe seven individuals affected with neurological symptoms who carry damaging heterozygous variants in IRF2BPL. Five cases carrying nonsense variants in IRF2BPL resulting in a premature stop codon display severe neurodevelopmental regression, hypotonia, progressive ataxia, seizures, and a lack of coordination. Two additional individuals, both with missense variants, display global developmental delay and seizures and a relatively milder phenotype than those with nonsense alleles. The bioinformatics signature for IRF2BPL based on population genomics is consistent with a gene that is intolerant to variation. We show that the IRF2BPL ortholog in the fruit fly, called pits (protein interacting with Ttk69 and Sin3A), is broadly expressed including the nervous system. Complete loss of pits is lethal early in development, whereas partial knock-down with RNA interference in neurons leads to neurodegeneration, revealing requirement for this gene in proper neuronal function and maintenance. The nonsense variants in IRF2BPL identified in patients behave as severe loss-of-function alleles in this model organism, while ectopic expression of the missense variants leads to a range of phenotypes. Taken together, IRF2BPL and pits are required in the nervous system in humans and flies, and their loss leads to a range of neurological phenotypes in both species.

genetics↗