Biology subjects

Becker, K. A.

Publications and source records attributed to Becker, K. A..

6 recordsLinked to original sources

bioRxiv2026

Analysis of Flurothyl-induced Seizures and Epileptogenesis in Mice with Targeted Deletions of Exons 3 and 4 in Dock7

Mutations in DOCK7 have been identified in individuals with epileptic encephalopathies. Given that epileptic encephalopathies are a set of disorders that result in seizure activity and associated cognitive and behavioral impairments, we investigated the role of Dock7 in seizure susceptibility and flurothyl kindling using the repeated flurothyl seizure model in mice. Male and female Dock7+/+ and Dock7{bigtriangleup}ex3-4/{bigtriangleup}ex3-4 mice were subjected to 8 daily flurothyl exposures (kindling, induction phase) followed by a 28-day incubation period and a subsequent flurothyl rechallenge (retest). No significant differences were observed in baseline myoclonic jerk or generalized seizure thresholds between genotypes or sexes. However, over the kindling period, male Dock7{bigtriangleup}ex3-4/{bigtriangleup}ex3-4 mice exhibited slightly higher myoclonic jerk and generalized seizure thresholds compared to Dock7+/+ males across trials. Female mice showed similar trends, but the differences were only significant for generalized seizure thresholds. Following the 28-day incubation period and flurothyl retest, male mice of both genotypes maintained their seizure thresholds upon retest. Dock7+/+ female mice showed increased myoclonic jerk and generalized seizure thresholds during retest, while Dock7{bigtriangleup}ex3-4/{bigtriangleup}ex3-4 females maintained their thresholds. A key finding was the emergence of more severe forebrain[->]brainstem seizures upon flurothyl retest in a significant percentage of mice across all groups. However, the proportion of mice developing these seizures did not differ significantly between genotypes. Although DOCK7 mutations have been linked to human epileptic encephalopathies and neurodevelopmental dysfunction, we find that Dock7{bigtriangleup}ex3-4/{bigtriangleup}ex3-4 male and female mice do not show heightened excitability or seizure susceptibilities using the repeated flurothyl seizure model. HighlightsO_LIDock7{bigtriangleup}ex3-4/{bigtriangleup}ex3-4 mice show slightly higher seizure thresholds during flurothyl kindling C_LIO_LIDock7{bigtriangleup}ex3-4/{bigtriangleup}ex3-4 mice do not exhibit heightened seizure susceptibility upon retest. C_LIO_LIForebrain-brainstem seizures emerged upon retest regardless of Dock7 genotype. C_LI

neuroscience↗

bioRxiv2026

Deletion of Dock7 Exons 3 and 4 Results in Reduced Trabecular Microarchitecture and a Decrease in Mineralization

Dedicator of Cytokinesis 7 (DOCK7) has recently emerged as a regulator of skeletal homeostasis, but existing Dock7 mutant models harbor only global mutations and are incompatible with tissue-specific deletion studies. We previously generated a Dock7-floxed allele in which exons 3-4 are flanked by LoxP sites. To validate the utility of this allele for future conditional strategies, we globally deleted exons 3-4 to generate Dock7em2/em2 mice and characterized their skeletal phenotype. Homozygous Dock7em2/em2 mice exhibited a diluted coat color and white belly spot, consistent with spontaneous Dock7 mutations, whereas heterozygotes exhibited no spotting or coat changes. Bone microarchitecture was assessed in 21-week-old males and females. Global deletion of Dock7 exons 3-4 resulted in a 30-37% reduction in trabecular bone volume in the distal femur and L5 vertebrae. Cortical bone thickness was unchanged, though sex-specific alterations in femoral area suggested altered appositional bone growth. Mass spectrometry (SWATH) analysis of Dock7em2/em2 bone marrow stromal cells (BMSCs) identified DOCK7 protein levels similar to control mice, likely resulting from an alternative translational start site. Despite stable protein abundance prior to differentiation, BMSCs from Dock7em2/em2 mice exhibited reduced mineralization and decreased Bglap expression, indicating attenuated osteoblast differentiation. These findings demonstrate that Dock7 exons 3-4 are required for normal trabecular bone acquisition and osteoblast function. While the Dock7-em2 allele likely encodes a truncated protein product, its recapitulation of the Misty skeletal phenotype confirms that exons 3-4 are essential for DOCK7 activity. These studies in the Dock7em2/em2 mouse provide a foundation for future tissue-specific deletion studies utilizing the Dock7 exon 3-4 deletion model to define the cellular roles of DOCK7 in regulating bone formation and trabecular architecture. Lay SummaryIdentifying genes that regulate bone mass is essential for understanding osteoporosis. Mutation of the Dock7 gene in mice results in low bone mass, similar to human osteoporosis. Previous models could not isolate its role in specific tissues, limiting our ability to understand how DOCK7 controls bone mass. We developed a new mouse model where Dock7 can be mutated in specific tissues. In this study, we bred mice to carry this mutation across all tissues, causing significant bone loss. This validates a flexible tool for future research to mutate DOCK7 in specific cell types and map its role in skeletal health.

cell biology↗

bioRxiv2025

DOCK7 regulates WNT signaling and NMJ clustering in normal and DMD muscle

Dedicator of cytokinesis (DOCK) proteins have diverse and critical roles in myoblast fusion, glucose metabolism, skeletal muscle regeneration, and neuronal polarity. DOCK7 is part of this 11-member family of atypical guanine exchange factors. While human DOCK7 pathogenic variants are rare, biallelic variants are known to result in hypotonia and ataxia in children. More recently, DOCK variants have been shown to impact disease pathogenesis as secondary modifiers. We used tissue-specific Dock7 conditional knockout mice to evaluate the roles of DOCK7 in the skeletal muscles and motor neurons. Both Dock7 knockout models developed significant nerve and muscle function deficits; however, the Dock7 motor neuron knockout mice showed more severe locomotor pathologies. RNA sequencing of the Dock7 knockout skeletal muscles revealed dysregulation of WNT signaling factors, and skeletal muscle repair and regeneration pathways. Molecular analysis revealed impaired DOCK7-mediated RAC1 activation that impacted acetylcholine receptor (AChR) clustering in both models. Interestingly, haploinsufficiency of Dock7 in mdx mice improved overall dystrophic muscle pathologies. We propose that DOCK7-RAC1 interactions result in activation of the WNT signaling pathway via the LRP4 receptor in the skeletal muscle and facilitate post-synaptic AChR clustering. Graphical AbstractDOCK7 is a regulator of normal and DMD muscle via the RAC1/WNT pathway. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/696254v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1ce84eaorg.highwire.dtl.DTLVardef@17855f1org.highwire.dtl.DTLVardef@15263d2org.highwire.dtl.DTLVardef@1778e0_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2022

Cell-intrinsic ceramides determine T cell function during melanoma progression

Acid sphingomyelinase (Asm) and acid ceramidase (Ac) are parts of the sphingolipid metabolism. Asm hydrolyzes sphingomyelin to ceramide, which is further metabolized to sphingosine by Ac. Ceramide generates ceramide-enriched platforms that are involved in receptor clustering within cellular membranes. However, the impact of cell-intrinsic ceramide on T cell function is not well characterized. By using T cell-specific Asm- or Ac-deficient mice, with reduced or elevated ceramide levels in T cells, we identified ceramide to play a crucial role in T cell function in vitro and in vivo. T cell-specific ablation of Asm in Asmflox/flox/CD4cre mice resulted in enhanced tumor progression associated with impaired T cell responses, whereas Acflox/flox/CD4cre mice showed reduced tumor growth rates and elevated T cell activation compared to the respective controls upon tumor transplantation. Further in vitro analysis revealed that decreased ceramide content supports CD4+ regulatory T cell differentiation and interferes with cytotoxic activity of CD8+ T cells. In contrast, elevated ceramide concentration in CD8+ T cells from Acflox/flox/CD4cre mice was associated with enhanced cytotoxic activity. Strikingly, ceramide co-localized with the T cell receptor (TCR) and CD3 in the membrane of stimulated T cells and phosphorylation of TCR signaling molecules was elevated in Ac-deficient T cells. Hence, our results indicate that modulation of ceramide levels, by interfering with the Asm or Ac activity has an effect on T cell differentiation and function and might therefore represent a novel therapeutic strategy for the treatment of T cell-dependent diseases such as tumorigenesis.

immunology↗

bioRxiv2021

Staphylococcus aureus α-toxin induces acid sphingomyelinase release from a human endothelial cell line

Staphylococcus aureus (S. aureus) is well known to express a plethora of toxins of which the pore-forming hemolysin A (-toxin) is the best-studied cytolysin. Pore-forming toxins (PFT) permeabilize host membranes during infection thereby causing concentration-dependent effects in host cell membranes ranging from disordered ion fluxes to cytolysis. Host cells possess defense mechanisms against PFT attack, resulting in endocytosis of the breached membrane area and delivery of repair vesicles to the insulted plasma membrane as well as a concurrent release of membrane repair enzymes. Since PFTs from several pathogens have been shown to recruit membrane repair components, we here investigated whether staphylococcal -toxin is able to induce these mechanisms in endothelial cells. We show that S. aureus -toxin induced increase in cytosolic Ca2+ in endothelial cells, which was accompanied by p38 MAPK phosphorylation. Toxin challenge led to increased endocytosis of an extracellular fluid phase marker as well as increased externalization of LAMP1-positive membranes suggesting that peripheral lysosomes are recruited to the insulted plasma membrane. We further observed that thereby the lysosomal protein acid sphingomyelinase (ASM) was released into the cell culture medium. Thus, our results show that staphylococcal -toxin triggers mechanisms in endothelial cells, which have been implicated in membrane repair after damage of other cell types by different toxins.

microbiology↗

bioRxiv2018

Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology

CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the methods success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA-2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.

genetics↗