Biology subjects

Watts, J. L.

Publications and source records attributed to Watts, J. L..

8 recordsLinked to original sources

bioRxiv2025

Gene replacement therapy for Piga GPI-anchor deficiency in the developing nervous system

Glycosylphosphatidylinositol (GPI) anchors are a class of post-translational modifications observed on over 150 proteins. Pathogenic variants in the GPI biosynthesis enzyme, PIGA, in humans are associated with several brain anomalies such as hypomyelination, cerebellar hypoplasia, ataxic gait, and can lead to premature mortality. We previously genetically deleted Piga from the embryonic mouse brain which led to early postnatal death and significant structural brain malformations similar to those observed in humans with PIGA variants. The current treatment options for PIGA patients only manage symptoms and provides palliative care, demonstrating a need for new therapeutic options. We employed an AAV9-mediated PIGA gene-replacement (AAV9-hPIGA) strategy to assess the efficacy of gene therapy in the brain. We show that a single intracerebroventricular treatment on the first day of life successfully rescued survival rates, structural brain anomalies, and neurological impairments. Additionally, we used mass spectrometry to identify and quantify GPI-anchored proteins in untreated and treated mutant mice. We found that AAV9- hPIGA treatment restored GPI-anchored protein levels in mutant animals. These investigations enhance our understanding of GPI-anchored protein production during brain development and contribute to the development of a more effective intervention for PIGA-related symptoms. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/693709v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@32038dorg.highwire.dtl.DTLVardef@1875eb5org.highwire.dtl.DTLVardef@5abfe8org.highwire.dtl.DTLVardef@1ed5719_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LIA single AAV9-hPIGA injection rescues Piga-related phenotypes including survival and structural brain defects C_LIO_LIAAV9-hPIGA treatment rescued long-term ataxia phenotypes in Piga mutants C_LIO_LINeuronal GPI-anchored protein levels in Piga mutants are increased with AAV9- hPIGA gene replacement C_LI

genetics↗

bioRxiv2025

Genetic Dissection of the role of Piga and Pgap2 in the embryonic mouse brain

Glycosylphosphatidylinositol (GPI) anchors are a post-translational modification of over 150 proteins. These GPI-anchored proteins are enriched in lipid rafts in the plasma membrane and serve a variety of functions including acting as receptors and cell adhesion molecules. Human pathogenic variants in GPI biosynthesis pathway enzymes are collectively called inherited GPI deficiencies and lead to several brain anomalies such as global developmental delay, hypomyelination, cerebellar hypoplasia, and premature death. PIGA is the first catalytic enzyme in the GPI anchor biosynthesis cascade, which creates the backbone of all GPI anchors, and PGAP2 is one of the last enzymes in the GPI-anchor biosynthesis pathway and modifies the protein-bound anchor for trafficking to the cell membrane. A Nestin-Cre mediated deletion of Piga in the mouse led to early postnatal death and significant structural brain malformations, similar to pathogenic human PIGA variants. While this model provides an opportunity to develop treatments for these neurological phenotypes, we still lack a deep understanding of GPI-anchor functions in brain development. Additionally, there are no studies on Pgap2 in the brain to date. We extended these studies with a series of genetic ablations to precisely determine the role of Piga and Pgap2 in the forebrain, oligodendrocytes, and cerebellum. We find Piga expression in the hindbrain is absolutely required for survival while Pgap2 ablations were much less deleterious. These studies broaden our knowledge on the brain region-specific requirements of GPI-anchor biosynthesis enzymes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/692975v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@18eb4feorg.highwire.dtl.DTLVardef@cb152org.highwire.dtl.DTLVardef@14d7e84org.highwire.dtl.DTLVardef@ad2d9a_HPS_FORMAT_FIGEXP M_FIG C_FIG Significance StatementIn this study, we examine the roles of the GPI-anchor biosynthesis enzymes Piga and Pgap2 to understand the poorly understood mechanisms underlying brain anomalies seen in the inherited GPI deficiencies. Using Cre/lox conditional mouse genetic models, we deleted Piga and Pgap2 from various brain regions, including the forebrain, oligodendrocytes, and cerebellum, and observed brain abnormalities. We find that Piga ablations mimic many brain phenotypes seen in human inherited GPI deficiencies, while Pgap2 ablations do not grossly disrupt neurodevelopment. Additionally, we demonstrate that Piga expression is essential in the cerebellum for survival. These findings reveal mechanisms underlying several inherited GPI deficiency brain phenotypes and will inform targeted therapeutic strategies.

genetics↗

bioRxiv2023

Does stomatal patterning in amphistomatous leavesminimize the CO2 diffusion path length withinleaves?

Photosynthesis is co-limited by multiple factors depending on the plant and its environment. These include biochemical rate limitations, internal and external water potentials, temperature, irradiance, and carbon dioxide (CO2). Amphistomatous leaves have stomata on both abaxial and adaxial leaf surfaces. This feature is considered an adaptation to alleviate CO2 diffusion limitations in productive environments where other factors are not limiting as the diffusion path length from stomate to chloroplast is effectively halved. Plants can also reduce CO2 limitations through other aspects of optimal stomatal anatomy: stomatal density, distribution, patterning, and size. A number of studies have demonstrated that stomata are overdispersed on a single leaf surface; however, much less is known about stomatal anatomy in amphistomatous leaves, especially the coordination between leaf surfaces, despite their prevelance in nature and near ubiquity among crop species. Here we use novel spatial statistics based on simulations and photosynthesis modeling to test hypotheses about how amphistomatous plants may optimize CO2 limitations in the model angiosperm Arabidopsis thaliana grown in different light environments. We find that 1) stomata are overdispersed, but not ideally dispersed, on both leaf surfaces across all light treatments; 2) abaxial and adaxial leaf surface patterning are independent; and 3) the theoretical improvements to photosynthesis from abaxial-adaxial stomatal coordination are miniscule (<< 1%) across the range of feasible parameter space. However, we also find that 4) stomatal size is correlated with the mesophyll volume that it supplies with CO2, suggesting that plants may optimize CO2 diffusion limitations through alternative pathways other than ideal, uniform stomatal spacing. We discuss the developmental, physical, and evolutionary constraits which may prohibit plants from reaching the theoretical adaptive peak of uniform stomatal spacing and inter-surface stomatal coordination. These findings contribute to our understanding of variation in the anatomy of amphistomatous leaves.

plant biology↗

bioRxiv2023

Dihydroxy-Metabolites of Dihomo-gamma-linolenic Acid Drive Ferroptosis- Mediated Neurodegeneration

Even after decades of research, the mechanism of neurodegeneration remains understudied, hindering the discovery of effective treatments for neurodegenerative diseases. Recent reports suggest that ferroptosis could be a novel therapeutic target for neurodegenerative diseases. While polyunsaturated fatty acid (PUFA) plays an important role in neurodegeneration and ferroptosis, how PUFAs may trigger these processes remains largely unknown. PUFA metabolites from cytochrome P450 and epoxide hydrolase metabolic pathways may modulate neurodegeneration. Here, we test the hypothesis that specific PUFAs regulate neurodegeneration through the action of their downstream metabolites by affecting ferroptosis. We find that the PUFA, dihomo gamma linolenic acid (DGLA), specifically induces ferroptosis-mediated neurodegeneration in dopaminergic neurons. Using synthetic chemical probes, targeted metabolomics, and genetic mutants, we show that DGLA triggers neurodegeneration upon conversion to dihydroxyeicosadienoic acid through the action of CYP-EH, representing a new class of lipid metabolite that induces neurodegeneration via ferroptosis.

neuroscience↗

bioRxiv2022

ATFS-1 regulates peroxisome assembly genes and protects both mitochondria and peroxisomes during peroxin perturbations

Peroxisome biogenesis disorders lead to a myriad of clinical manifestations, among which is the dysfunction of the mitochondria. Mitochondria dysfunction is typically sensed by the UPRmt, a broad protective transcriptional response governed by the transcription factor ATFS-1. Here, we investigated the role of the UPRmt during peroxisomal stress. We show that mutations or knockdown of peroxins, the genes required for peroxisome assembly, lead to mitochondria dysfunction and the induction of the UPRmt in C.elegans. The UPRmt induced the transcription of the mitochondrial outer membrane translocase mspn-1 (ATAD-1), that in turn alleviated mitochondrial stress most likely by extracting mislocalized proteins. Importantly, ATFS-1 regulated the transcription of peroxins and the peroxisomal transporter pmp-4. A prx-5 loss of function strain induced a retrograde response that resulted in the transcriptional induction of peroxins, peroxisomal transporters, chaperons and proteases. And was dependent on ATFS-1 and on the peroxisome proliferator activator receptor alpha, NHR-49. Knockout of atfs-1 during peroxisomal stress resulted in severe developmental delays, import defects to peroxisomes and the appearance of large peroxisomal structures. We propose that ATFS-1 regulates the biogenesis of peroxisomes and protects the organism by alleviating the stress of both peroxisomes and mitochondria during peroxisomal stress.

developmental biology↗

bioRxiv2019

Dietary Induction and Modulation of Ferroptosis in Caenorhabditis elegans

Ferroptosis is an iron-dependent form of regulated cell death associated with oxidized polyunsaturated phospholipids. Understanding the role of this process in vivo has been slowed by the lack of readily accessible model systems. Exposing the nematode Caenorhabditis elegans to the polyunsaturated fatty acid dihomogamma-linolenic acid (DGLA; 20:3n-6) causes germ cell death and sterility that is largely independent of the canonical apoptosis pathway. Here we demonstrate that DGLA-induced germ cell death is modulated by small molecule ferroptosis inhibitors, genetic manipulation of ferritin, NADPH oxidase, and glutathione peroxidases, and by dietary co-supplementation with oleic acid. Thus, DGLA-induced germ cell death in C. elegans is highly analogous to ferroptosis in mammalian cells. DGLA can also induce ferroptosis in human cells, further highlighting this omega-6 PUFA as a metabolic instigator of ferroptosis. Together, these results establish C. elegans as a powerful animal model to study the induction and modulation of ferroptosis by dietary fats.\n\nHighlights- Dietary dihomogamma-linolenic acid (DGLA)-induced germ cell death in C. elegans is alleviated by small molecule antioxidants and iron chelators\n- Dietary and endogenous oleic acid protects from DGLA-induced ferroptosis\n- Ether-lipid deficiency increases sensitivity to DGLA-induced ferroptosis\n- DGLA specifically induces ferroptosis in human cancer cells

cell biology↗

bioRxiv2019

TEAD4/YAP1/WWTR1 prevent the premature onset of pluripotency prior to the 16-cell stage

In the mouse embryo, pluripotent cells arise inside the embryo around the 16-cell stage. During these early stages, Sox2 is the only gene whose expression is known to be induced specifically within inside cells as they are established. To understand how pluripotent cells are created, we investigated the mechanisms regulating the initial activation of Sox2 expression. Surprisingly, Sox2 expression initiated normally in the absence of both Nanog and Oct4, highlighting differences between embryo and stem cell models of pluripotency. However, we observed precocious, ectopic expression of Sox2 prior to the 16-cell stage in the absence of Yap1, Wwtr1, and Tead4. Interestingly, the repression of premature Sox2 expression was sensitive to LATS1/2 activity, even though it normally does not limit TEAD4/YAP1/WWTR1 activity during these early stages. Finally, we present evidence for direct transcriptional repression of Sox2 by YAP1/WWTR1/TEAD4. Taken together, our observations reveal that, while embryos are initially competent to express Sox2 as early as the 4-cell stage, transcriptional repression prevents the premature expression of Sox2, thereby restricting the pluripotency program to the stage when inside cells are first created.

developmental biology↗

bioRxiv2018

Previously characterized Caenorhabditis elegans RNA interference-resistant mutants retain substantial silencing capacity

RNA interference is a powerful tool for dissecting gene function. In Caenorhabditis elegans, ingestion of double stranded RNA causes strong, systemic knockdown of target genes. Further insight into gene function can be revealed by tissue-specific RNAi techniques. Currently available tissue-specific C. elegans strains rely on rescue of RNAi function in a desired tissue or cell in an otherwise RNAi deficient genetic background. We attempted to assess the contribution of specific tissues to polyunsaturated fatty acid (PUFA) synthesis using currently available tissue-specific RNAi strains. We discovered that rde-1 (ne219), a commonly used RNAi-resistant mutant strain, retains considerable RNAi capacity against RNAi directed at PUFA synthesis genes. By measuring changes in the fatty acid products of the desaturase enzymes that synthesize PUFAs, we found that the before mentioned strain, rde-1 (ne219) and the reported germline only RNAi strain, rrf-1 (pk1417) are not appropriate genetic backgrounds for tissue-specific RNAi experiments. However, the knockout mutant rde-1 (ne300) was strongly resistant to dsRNA induced RNAi, and thus is more appropriate for construction of a robust tissue-specific RNAi strains. Using newly constructed strains in the rde-1(null) background, we found considerable desaturase activity in intestinal, epidermal, and germline tissues, but not in muscle. The RNAi-specific strains reported in this study will be useful tools for C. elegans researchers studying a variety of biological processes.

molecular biology↗