Biology subjects

Snyder, K. M.

Publications and source records attributed to Snyder, K. M..

4 recordsLinked to original sources

bioRxiv2022

Ablation of SYK kinase from primary human Natural Killer cells via CRISPR/Cas9 enhances cytotoxicity and cytokine production

Cytomegalovirus (CMV) infection alters natural killer (NK) cell phenotype and function toward a more memory-like immune state. These cells, termed adaptive NK cells, typically express CD57 and NKG2C but lack expression of the Fc receptor {gamma} chain (Gene: FCER1G, FcR{gamma}), PLZF, and SYK. Functionally, adaptive NK cells display enhanced antibody-dependent cellular cytotoxicity (ADCC) and cytokine production. However, the mechanism behind this enhanced function is unknown. To understand what drives cytotoxicity and cytokine production in adaptive NK cells, we optimized a CRISPR/Cas9 system to ablate genes from primary human NK cells. ADCC by human NK cells is exclusively mediated by the CD16A (Fc{gamma}RIIIA) signaling apparatus, which includes FcR{gamma}, CD3{zeta}, SYK, SHP-1, ZAP-70, and the transcription factor PLZF. We ablated the genes encoding these molecules and tested subsequent ADCC and cytokine production. We found that ablating the FcR{gamma} chain caused a modest increase in TNF production. Ablation of PLZF did not enhance ADCC or cytokine production. Importantly, SYK kinase ablation significantly enhanced both cytotoxicity and cytokine production, while ZAP-70 kinase ablation diminished function. Ablation of the phosphatase SHP-1 resulted in mixed effects on function, with NK cells demonstrating enhanced cytotoxicity but reduced cytokine production. These results indicate that the enhanced cytotoxicity and cytokine production of CMV-induced adaptive NK cells is more likely due to the loss of SYK than the lack of FcR{gamma} or PLZF. The lack of SYK expression may limit SHP-1-mediated inhibition of CD16A signaling, leading to enhanced cytotoxicity and cytokine production. In addition to providing mechanistic answers about CMV-induced adaptive NK cell functionality, our results indicate that NK chimeric antigen receptor (CAR) therapeutics that invoke ADCC signaling molecules (e.g., CD3{zeta} chain) may benefit from ablating SYK, while maintaining ZAP-70, to increase functionality.

immunology↗

bioRxiv2021

The critical role of the ventral temporal lobe in word retrieval

Deficits in word retrieval are a hallmark of a variety of neurological illnesses spanning from dementia to traumatic injuries. The role of the dominant temporal lobe in fluent naming has been characterized by lesional analyses, functional imaging, and intracranial recordings, but limitations of each of these measures preclude a clear assessment of which specific constituent of the temporal lobe is critical for naming. We studied a large cohort of patients undergoing surgical resections or laser ablations of the dominant temporal lobe for medically intractable epilepsy (n=95). These techniques are exceedingly effective for seizure control but often result in language declines, particularly in confrontation naming, which can be socio-economically disabling. We used a multivariate voxel-based lesion symptom mapping analysis to localize brain regions significantly associated with visual object naming deficits. We observed that posterior inferior temporal regions, centered around the middle fusiform gyrus, were significantly associated with a decline in confrontation naming. Furthermore, we found that the posterior margin of anterior temporal lobectomies was linearly correlated to a decline in visual naming with a clinically significant decline occurring once the resection extended 6 cm from the anterior tip of the temporal lobe. We integrated these findings with electrocorticography during naming in a subset of this population and found that the majority of cortical regions whose resection was associated with a significant decline overlapped with regions that were functionally most active prior to articulation. Importantly, these loci coincide with the sites of susceptibility artifacts during echo-planar imaging, which explains why this region has not previously been implicated. Taken together, these data highlight the crucial contribution of the posterior ventral temporal cortex in lexical access and its important role in the pathophysiology of anomia following temporal lobe resections. Surgical strategies, including the use of laser ablation to target the medial temporal lobe as well as microsurgical approaches, should attempt to preserve this region to mitigate postoperative language deficits.

neuroscience↗

bioRxiv2020

miRNA-mediated loss of m6A increases nascent translation in glioblastoma

Within the glioblastoma cellular niche, glioma stem cells (GSCs) can give rise to differentiated glioma cells (DGCs) and, when necessary, DGCs can reciprocally give rise to GSCs to maintain the cellular equilibrium necessary for optimal tumor growth. Here, using ribosome profiling, transcriptome and m6A RNA sequencing, we show that GSCs from patients with different subtypes of glioblastoma share a set of transcripts, which exhibit a pattern of m6A loss and increased protein translation during differentiation. The target sequences of a group of miRNAs overlap the canonical RRACH m6A motifs of these transcripts, many of which confer a survival advantage in glioblastoma. Ectopic expression of the RRACH-binding miR-145 induces loss of m6A, formation of FTO/AGO1/ILF3/miR-145 complexes on a clinically relevant tumor suppressor gene (CLIP3) and significant increase in its nascent translation. Inhibition of miR-145 maintains RRACH m6A levels of CLIP3 and inhibits its nascent translation. This study highlights a critical role of miRNAs in assembling complexes for m6A demethylation and induction of protein translation during GSC state transition. Author SummaryCellular plasticity and epigenetic adaptation of human glioblastoma stem cells to the tumor microenvironment is a hallmark of this devastating disease. With our present work, we discover the relationship between miRNAs and the RNA methylation machinery in human glioblastoma and show how miRNA-induced loss of m6A results in increase in protein translation of clinically important transcripts during glioblastoma stem cell differentiation. Leveraging the dynamic functions of these miRNAs can be important in the design of optimal therapeutics targeted at cancer cell plasticity.

cancer biology↗

bioRxiv2020

Ectodomain shedding of L-selectin by ADAM17 in canine neutrophils

The adhesion protein L-selectin (CD62L) is expressed at high levels by circulating neutrophils and has a critical role in initiating their recruitment at sites of inflammation. L-selectin expression is rapidly downregulated upon neutrophil activation by various stimuli through a proteolytic process referred to as ectodomain shedding, which regulates L-selectins binding avidity and neutrophil recruitment. In humans and mice, L-selectin shedding is primarily mediated by ADAM17 (a disintegrin and metalloproteinase 17). L-selectin expression is also rapidly downregulated by canine neutrophils upon their activation; however, the role of ADAM17 in this process has not been previously investigated. We show that a highly selective inhibitor of ADAM17, but not an inhibitor of its most closely related family member ADAM10, effectively blocked L-selectin downregulation from the surface of canine neutrophils following their activation. The ADAM17 inhibitors did not block the rapid upregulation of the CD18 integrin Mac-1 (CD11b/CD18), showing that they did not broadly impair neutrophil activation. To directly examine the expression of ADAM17, we used several anti-human ADAM17 mAbs. Many did not stain canine neutrophils; however, the ADAM17 function-blocking mAbs MEDI3622 and D1(A12) did stain and also blocked L-selectin downregulation. Taken together, our findings provide the first direct evidence that ADAM17 is a primary sheddase of canine L-selectin, which may serve as an important therapeutic target to prevent neutrophil dysfunction during conditions of excessive inflammation, such as sepsis.

immunology↗