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Walker, M. A.

Publications and source records attributed to Walker, M. A..

8 recordsLinked to original sources

A DNM1L GTPase Effector Domain Deletion Causes Intermediate Defects in Peroxisomal and Mitochondrial Dynamics and Reveals a Targetable Metabolic Vulnerability

DNM1L encodes dynamin-related protein 1 (DRP1), a key GTPase regulating mitochondrial and peroxisomal dynamics. Pathogenic DNM1L variants are associated with a broad spectrum of neurodevelopmental and neurodegenerative disorders, with phenotypic severity varying by affected protein domain. While variants in the GTPase domain are typically associated with milder phenotypes and middle-domain variants with severe developmental and epileptic encephalopathy, the functional impact of GTPase effector domain (GED) variants remains less defined. Here, we describe four individuals harboring a recurrent heterozygous GED deletion (L700del) presenting with movement disorders and lacking severe infantile encephalopathy, consistent with an intermediate clinical phenotype. Patient-derived fibroblasts exhibited tubular peroxisomes and enlarged mitochondria, while Drosophila models demonstrated that DNM1LL700del has intermediate functional effects that were more pronounced than those of a GTPase-domain variant A192E but less severe than those of the middle-domain variant G350R. Comparison with another GED variant, Y691C, further demonstrated variant-specific differences in severity within the GED. Metabolic stress assays showed that L700del fibroblasts exhibited impaired growth under galactose conditions, consistent with increased vulnerability under conditions dependent on mitochondrial oxidative metabolism. Bezafibrate, a PPAR agonist that promotes mitochondrial biogenesis, partially rescued this growth defect. Together, these findings support a functional allelic spectrum in DNM1L-associated disease in which protein domain contributes to, but does not completely determine, functional severity. L700del represents an intermediate-strength allele associated with a movement-disorder phenotype and a pharmacologically modifiable metabolic vulnerability.

genetics↗

4,5-dihydroxyhexanoic acid is a robust circulating and urine marker of mitochondrial disease and its severity

Management of patients with mitochondrial respiratory chain diseases is challenging, in part because of our incomplete understanding of pathogenesis and a lack of biomarkers. Unknown metabolites account for >90% of detected features in modern metabolomics experiments and hold immense untapped promise for new basic and biomedical research. We recently used mass spectrometry-based metabolomics to identify and validate 19 circulating blood-based biomarkers for patients with the mitochondrial DNA (mtDNA) m.3243A>G pathogenic variant, which is the most frequent cause of the mitochondrial disorder MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). However, the most significantly changing biomarker corresponded to an "unknown" metabolite. Here, we combine cheminformatics with analytical chemistry and identify that feature as 4,5-dihydroxyhexanoic acid (4,5-DHHA), a metabolite previously associated with inherited defects of gamma-aminobutyric acid (GABA) catabolism, but with no prior links to mitochondrial respiratory chain disorders. We validate this finding in an independent MELAS cohort and further show that 4,5-DHHA levels correlate with disease severity and are elevated in patients with other forms of mitochondrial disease and sepsis. Furthermore, brain 4,5-DHHA levels were elevated in two genetic mouse models of mitochondrial disease. In vitro and tissue culture experiments indicate that 4,5-DHHA is generated when the GABA catabolite succinic semialdehyde reacts with an intermediate of the pyruvate dehydrogenase reaction and is sensitive to mitochondrial complex I function. Our work identifies 4,5-DHHA as a robust plasma and urine marker of mitochondrial dysfunction in humans and reveals new connections between the respiratory chain and GABA metabolism. Significance StatementInborn errors of the mitochondrial respiratory chain cause severe, progressive diseases, yet effective treatments and biomarkers remain limited. Modern metabolomics detects thousands of molecules in biofluids, but the vast majority are unidentified. In this study, we investigate the most significantly altered blood metabolite in patients with the most common mitochondrial disease - MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) - and identify it as an 4,5-dihydroxyhexanoic acid (4,5-DHHA). We show that 4,5-DHHA is reproducibly elevated and correlates with severity. Levels are increased across multiple mitochondrial disorders as well as in sepsis and rise when respiratory chain function is impaired. These findings establish 4,5-DHHA as a promising biomarker of mitochondrial dysfunction and reveal a link to dysregulated GABA metabolism.

biochemistry↗

Exploratory Assessment of Preconception Phthalate Exposure on Fertility and Offspring Health in Mice

Infertility affects 10-15% of couples worldwide and is increasingly attributed to environmental exposures, particularly endocrine-disrupting chemicals (EDCs) such as phthalates. While gestational exposures are well studied, little is known about how preconception exposures influence fertility and offspring health. Phthalate exposure altered estrous cyclicity, with dams spending more time in proestrus and less in metestrus but did not significantly impact implantation or litter size. At E14.5, exposed fetuses exhibited increased bodyweights, accompanied by an expansion of the placental junctional zone in males. Altered bodyweight persisted into adulthood, however adulthood offspring displayed a reduction in bodyweight. RNA-sequencing revealed widespread transcriptional reprogramming in female placentas (518 DEGs) affecting immune regulation, steroid metabolism, and extracellular matrix remodeling, while male placentas exhibited only 9 DEGs but showed structural alterations. In offspring livers, transcriptomic shifts were sex-specific: females displayed downregulation of metabolic and immune genes (e.g., Cyp7a1, Mthfr, H2-DMB1), while males showed upregulation of immune and signaling genes (e.g., Elf4, Adm2, Ly6a). Collectively, these findings demonstrate that preconception phthalate exposure induces subtle but biologically meaningful maternal endocrine disruption, alters placental structure and function, and reprograms offspring growth and liver transcriptomes in a sex-specific manner. This work identifies the preconception window as a critical period of vulnerability for EDC impacts on reproductive success and intergenerational health.

pharmacology and toxicology↗

Integration of diploid T2T parental genomes with progeny genomes and transcriptomes identifies resistance genes in the grape powdery mildew resistance loci Ren6 and Ren7 of Vitis piazeskii

The Chinese grape accession Vitis piasezkii DVIT2027 carries two loci associated with powdery mildew (PM) resistance loci, Ren6 and Ren7, which differ in timing and strength of response to Erysiphe necator. Both loci are consistent with recognition by intracellular immune receptors. To identify the underlying nucleotide-binding leucine-rich repeat (NLR) genes, we assembled chromosome-scale diploid genomes of DVIT2027 and the susceptible V. vinifera F2-35, parents of a segregating F1 population. We integrated these assemblies with deep resequencing data from eight F1 sib-lines carrying different Ren6/Ren7 combinations and generated trio-binned, parent-phased genomes for six progeny. This resolved both PM-resistant (PMR) and PM-susceptible (PMS) haplotypes at Ren6 and Ren7. Comparative analyses revealed extensive structural variation and complete haplotype specificity among NLRs, with several candidate genes lacking allelic counterparts in PMS haplotypes. Expression profiling across PMR sib-lines identified four and two CC-NBS-LRR genes potentially associated with Ren6 and Ren7, respectively. Sequence graph reconstruction of these loci across multiple V. piasezkii accessions revealed broad intraspecific diversity and DVIT2027-specific nodes, including within candidate NLR genes. These results provide a high-resolution view of Ren6 and Ren7 and support the identification of resistance gene candidates for functional validation and grapevine breeding.

plant biology↗

The grape powdery mildew resistance loci Ren2, Ren3, Ren4D, Ren4U, Run1, Run1.2b, Run2.1, and Run2.2 activate different transcriptional responses to Erysiphe necator

Multiple grape powdery mildew (PM) genetic resistance (R) loci have been found in wild grape species. Little is known about the defense responses associated with each R locus. In this study, we compare the defense mechanisms associated with PM resistance in interspecific crosses segregating for a single R locus from Muscadinia rotundifolia (Run1, Run1.2b, Run2.1, Run2.2), Vitis cinerea (Ren2), V. romanetii (Ren4D and Ren4U), and the interspecific hybrid Villard blanc (Ren3). By combining optical microscopy, visual scoring, and biomass estimation, we show that the eight R loci confer resistance by limiting infection at different stages. We assessed the defense mechanisms triggered in response to PM at 1 and 5 days post inoculation (dpi) via RNA sequencing. To account for the genetic differences between species, we developed for each accession a diploid synthetic reference transcriptome by incorporating into the PN40024 reference homozygous and heterozygous sequence variants and de novo assembled transcripts. Most of the R loci exhibited a higher number of differentially expressed genes (DEGs) associated with PM resistance at 1 dpi compared to 5 dpi, suggesting that PM resistance is mostly associated with an early transcriptional reprogramming. Comparison of the PM resistance-associated DEGs showed a limited overlap between pairs of R loci, and nearly half of the DEGs were specific to a single R locus. The largest overlap of PM resistance-associated DEGs was found between Ren3+, Ren4D+, and Ren4U+ genotypes at 1 dpi, and between Ren4U+ and Run1+ accessions at 5 dpi. The Ren3+, Ren4D+, and Ren4U+ were also found to have the highest number of R locus-specific DEGs in response to PM. Both shared and R locus-specific DEGs included genes from different defense-related categories, indicating that the presence of E. necator triggered distinct transcriptional responses in the eight R loci.

genetics↗

Characterization of the grape powdery mildew genetic resistance loci in Muscadinia rotundifolia Trayshed

Muscadinia rotundifolia cv. Trayshed is a valuable source of resistance to grape powdery mildew. It carries two powdery mildew resistance-associated genetic loci, Run1.2 on chromosome 12 and Run2.2 on chromosome 18. In this study, we identified, phased, and reconstructed the two haplotypes of each resistance-associated locus. Haplotype phasing allowed the identification of several structural variation events between haplotypes of both loci. Combined with manual refinement of the gene models, we found that the heterozygous structural variants affected the gene content, with some resulting in duplicated or hemizygous nucleotide-binding leucine-rich repeat (NLR) genes. Structural variations also impacted the domain composition of some NLRs. These findings emphasize the need of generating haplotype-resolved sequences instead of using consensus sequences for identifying haplotype-specific candidate genes. Comparison of the NLRs in the Run1.2 and Run2.2 loci indicated that the two loci are composed of a different number and classes of NLR genes. We provide a list of candidate NLR genes from the Run1.2b and Run2.2 loci, whose expression suggests a role in powdery mildew resistance in Trayshed. These first complete and haplotype-resolved resistance-associated loci, and their candidate NLR genes, represent new resources to develop powdery mildew-resistant grape cultivars.

plant biology↗

Mitochondrial interactome quantitation reveals structural changes in metabolic machinery in failing murine heart

Advancements of cross-linking mass spectrometry (XL-MS) for structural analysis of proteins bridges the gap between purified systems and native tissue environments. Here, isobaric quantitative protein interaction reporter technology (iqPIR) was utilized to further extend XL-MS to the first system-wide comparative study of mitochondrial proteins from healthy and diseased murine hearts. The failing heart interactome includes 602 statistically significant cross-linked peptide pairs altered in the disease condition. Structural insight into ketone oxidation metabolons, OXPHOS machinery, and nucleotide transporter hybrid-conformations, support mitochondrial remodeling in failing heart while bringing forth new hypotheses for pathological mechanisms. Application of quantitative cross-linking technology in tissue provides molecular-level insight to complex biological systems difficult to model in cell culture, thus providing a valuable resource for study of human diseases.

systems biology↗

The genomic diversification of clonally propagated grapevines

Vegetatively propagated clones accumulate somatic mutations. The purpose of this study was to better understand the consequences of clonal propagation and involved defining the nature of somatic mutations throughout the genome. Fifteen Zinfandel winegrape clone genomes were sequenced and compared to one another using a highly contiguous genome reference produced from one of the clones, Zinfandel 03.\n\nThough most heterozygous variants were shared, somatic mutations accumulated in individual and subsets of clones. Overall, heterozygous mutations were most frequent in intergenic space and more frequent in introns than exons. A significantly larger percentage of CpG, CHG, and CHH sites in repetitive intergenic space experienced transition mutations than genic and non-repetitive intergenic spaces, likely because of higher levels of methylation in the region and the increased likelihood of methylated cytosines to spontaneously deaminate. Of the minority of mutations that occurred in exons, larger proportions of these were putatively deleterious when they occurred in relatively few clones.\n\nThese data support three major conclusions. First, repetitive intergenic space is a major driver of clone genome diversification. Second, clonal propagation is associated with the accumulation of putatively deleterious mutations. Third, the data suggest selection against deleterious variants in coding regions such that mutations are less frequent in coding than noncoding regions of the genome.

genomics↗