Biology subjects

Johnson, K. J.

Publications and source records attributed to Johnson, K. J..

9 recordsLinked to original sources

bioRxiv2025

Your Brain Doesn't Look a Day Past 70! Cross-Sectional Associations with Brain-Predicted Age in the Cognitively-Intact Oldest-Old

The cognitively-intact oldest-old (85+) may be the most-resilient members of their birth cohort; due to survivorship effects (e.g., depletion of susceptibles), risk factors associated with brain aging biomarkers in younger samples may not generalize to the oldest-old. We evaluated associations between established aging-related risk factors and brain-predicted age difference (brainPAD) in a cross-sectional cognitively-intact oldest-old sample. Additionally, we evaluated brainPAD-cognition associations to characterize brain maintenance vs. cognitive reserve in our sample. Oldest-old adults (N = 206; 85-99 years; MoCA > 22 or neurologist evaluation) underwent T1-weighted MRI; brainPAD was generated with brainageR, such that more-positive brainPAD reflected relatively advanced brain aging. Sex, educational attainment, alcohol and smoking history, exercise history, BMI, cardiovascular and metabolic disease history, and anticholinergic medication burden were self-reported. Global cognitive z-score and coefficient of variation were derived from the NACC UDS 3.0 cognitive battery; crystallized-fluid discrepancy was derived from the NIH Toolbox Cognitive Battery. Mean brainPAD was -7.99 (SD: 5.37; range: -24.50, 6.03). Women showed more-delayed brain aging than men (B = -2.35, 95% CI = - 4.28, -0.41, p = 0.018). No other exposures were associated with brainPAD. BrainPAD was not associated with any cognitive variable. These findings suggest that cognitively-intact oldest-old adults may be atypically-resistant to risk factors associated with aging in younger samples, consistent with survivorship effects in aging. Furthermore, brainPAD may have limited explanatory value for cognitive performance in cognitively-intact oldest-old adults, potentially due to high cognitive reserve. Overall, our findings highlight the impact of survivorship effects on brain aging research. HighlightsO_LIBrain-predicted age difference was assessed in cognitively-intact oldest-old ([≥]85) C_LIO_LIMean brain-predicted age corresponded to an 8-year delay in brain aging C_LIO_LIBrain age in oldest-old was not associated with self-reported health history C_LIO_LIBrain age was not associated with cognitive performance C_LI

neuroscience↗

bioRxiv2024

Resistance to Naive and Formative Pluripotency Conversion in RSeT Human Embryonic Stem Cells

One of the most important properties of human embryonic stem cells (hESCs) is related to their primed and naive pluripotent states. Our previous meta-analysis indicates the existence of heterogeneous pluripotent states derived from diverse naive protocols. In this study, we have characterized a commercial medium (RSeT)-based pluripotent state under various growth conditions. Notably, RSeT hESCs can circumvent hypoxic growth conditions as required by naive hESCs, in which some RSeT cells (e.g., H1 cells) exhibit much lower single cell plating efficiency, having altered or much retarded cell growth under both normoxia and hypoxia. Evidently, hPSCs lack many transcriptomic hallmarks of naive and formative pluripotency (a phase between naive and primed states). Integrative transcriptome analysis suggests our primed and RSeT hESCs are close to the early stage of post-implantation embryos, similar to the previously reported primary hESCs and early hESC cultures. Moreover, RSeT hESCs did not express naive surface markers such as CD75, SUSD2, and CD130 at a significant level. Biochemically, RSeT hESCs exhibit a differential dependency of FGF2 and co-independency of both Janus kinase (JAK) and TGF{beta} signaling in a cell-line-specific manner. Thus, RSeT hESCs represent a previously unrecognized pluripotent state downstream of formative pluripotency. Our data suggest that human naive pluripotent potentials may be restricted in RSeT medium. Hence, this study provides new insights into pluripotent state transitions in vitro.

cell biology↗

bioRxiv2023

RAPID RESISTANCE TO BET INHIBITORS IS MEDIATED BY FGFR1 IN GLIOBLASTOMA

Bromodomain and extra-terminal domain (BET) proteins are therapeutic targets in several cancers including the most common malignant adult brain tumor glioblastoma (GBM). Multiple small molecule inhibitors of BET proteins have been utilized in preclinical and clinical studies. Unfortunately, BET inhibitors have not shown efficacy in clinical trials enrolling GBM patients. One possible reason for this may stem from resistance mechanisms that arise after prolonged treatment within a clinical setting. However, the mechanisms and timeframe of resistance to BET inhibitors in GBM is not known. To identify the temporal order of resistance mechanisms in GBM we performed quantitative proteomics using multiplex-inhibitor bead mass spectrometry and demonstrated that intrinsic resistance to BET inhibitors in GBM treatment occurs rapidly within hours and involves the fibroblast growth factor receptor 1 (FGFR1) protein. Additionally, small molecule inhibition of BET proteins and FGFR1 simultaneously induces synergy in reducing GBM tumor growth in vitro and in vivo. Further, FGFR1 knockdown synergizes with BET inhibitor mediated reduction of GBM cell proliferation. Collectively, our studies suggest that co-targeting BET and FGFR1 may dampen resistance mechanisms to yield a clinical response in GBM.

cancer biology↗

bioRxiv2023

Microglial depletion worsens lesion in female but not male C57BL/6J mice after P10 hypoxia-ischemia

BackgroundRodent models for perinatal hypoxic ischemic (HI) encephalopathy have reported sex differences such as the same injury causing larger lesions in the brains of males compared to females. Microglia, the resident immune cells of the brain that have sex-dependent developmental trajectories and gene expression patterns, likely play a different role in females and males following HI. However, there is conflicting literature on whether depletion of microglia worsens or improves HI-induced lesions and whether this differs by sex. Here we tested the effect of pharmacologic microglia depletion on HI lesion size in male and female mice. MethodsAn initial cohort of C57BL/6J mouse pups underwent HI at postnatal day 10 (P10) using a modified Vannucci procedure or a Sham insult followed by brain collection at P13. Another cohort of mice received daily intraperitoneal injections from P7 to P12 of either 25mg/kg PLX3397 (PLX, a CSF1R inhibitor) or vehicle (Veh). These mice also underwent HI or Sham at P10, resulting in four groups (Veh-Sham, Veh-HI, PLX-Sham, PLX-HI). All groups included female and male mice. Behavioral testing was performed both pre-HI (forelimb grasping [P8, P9]) and post-HI (open field traversal [P12], behavior and appearance observations [P13]). P13 brain sections underwent immunohistochemistry for Iba1 or cresyl violet staining for lesion scoring. ResultsP13 HI hippocampal sections had more Iba1 signal than Sham, with more variance in Male-HI vs Female-HI mice. PLX led to >95% depletion of Iba1+ cells at P10 or P13, and effective elimination of microglia did not differ by sex. In the hippocampus, Female-PLX-HI mice had worse lesion scores than Female-Veh-HI mice; this was not true in male mice, where there was a trend in the opposite direction. Female-PLX-HI mice also had worse lesion scores than Male-PLX-HI mice. In contrast to this sex-dependent effect of PLX on lesion score, there was no difference among groups in developmental milestones. ConclusionPLX3397 injection P7-P9 or P7-P12 effectively depletes microglia by P10 or P13, respectively. Microglial depletion via PLX worsens HI-induced injury in female mice but not in male mice.

neuroscience↗

bioRxiv2023

"Surviving and Thriving": Evidence for Cortical GABA Stabilization in Cognitively-Intact Oldest-Old Adults

Cortical GABA levels are reduced in older age; age-related differences in GABA may be associated with age-related cognitive change. The nature of age-related GABA differences in the highest-functioning stratum of the oldest-old (85+) population is not yet known. We extend our previously-reported Individual Participant Data Meta-Analysis of GABA levels (Porges et al., 2021) across the lifespan with four novel datasets sampling the cognitively-intact oldest-old. The slope of age-related GABA differences in cognitively-intact oldest-old adults flattens after roughly age 80. We interpret these findings as an effect of survivorship: inclusion in the study required intact cognition, and too great a reduction of GABA levels may not be compatible with neurophysiological function needed for intact cognition. This work contributes to a growing body of evidence suggesting that successful cognitive aging may require intact GABAergic function, as well as further characterizing successful aging amongst oldest-old adults.

neuroscience↗

bioRxiv2021

Evaluation of spontaneous seizure activity, sex-dependent differences, behavioral comorbidities, and alterations in CA1 neuron firing properties in a mouse model of Dravet Syndrome

Dravet syndrome (DS) is a rare childhood epilepsy disorder resulting in spontaneous, recurrent seizures (SRS) and behavioral co-morbidities. To facilitate the discovery and development of anti-seizure drugs for DS, the contract site of the NINDS Epilepsy Therapy Screening Program (ETSP) has continued to evaluate a mouse model of DS. Scn1aA1783V/WT mice exhibited increased hyperactivity, thigmotaxis, and deficits in nest-building behavior. Ex-vivo brain slice electrophysiology experiments revealed increased excitability of hippocampal CA1 neurons specifically due to increased action potential firing frequency in response to brief depolarizations and decreased frequency of spontaneous GABAergic synaptic events. A video-EEG study revealed mice had on average, 1 seizure per day, with males seizing significantly more frequently than females. Increased proportion of seizure activity occurred during the dark phase of the light/dark cycle in both sexes. While clobazam, a drug commonly prescribed to patients with DS, had no effect on SRS activity at the tested doses, the seizure history and frequency observed in this study aids in determining the sample sizes and experimental timeline needed for adequately powered preclinical drug studies. Overall, this study provides a broad description of the Scn1aA1783V/WT mouse and highlights the utility of this model in therapy discovery.

pharmacology and toxicology↗

bioRxiv2020

Development of an Antiseizure Drug Screening Platform for Dravet Syndrome at the NINDS contract site for the Epilepsy Therapy Screening Program

ObjectiveDravet syndrome (DS) is a rare, but catastrophic genetic epilepsy, with 80% of patients with carrying a mutation in the SCN1A gene. Currently, no anti-seizure drug (ASD) exists that adequately controls seizures. Patients with DS often present clinically with a febrile seizure and generalized tonic-clonic seizures that continue throughout life. To facilitate the development of ASDs for DS, the contract site of the NINDS Epilepsy Therapy Screening Program (ETSP) has evaluated a mouse model of DS using the conditional knock-in Scn1aA1783V/WT mouse. MethodsSurvival rates and temperature thresholds for Scn1aA1783V/WT were determined. Prototype ASDs were administered via intraperitoneal injections at the time-to-peak effect, which was previously determined, prior to the induction of hyperthermia-induced seizures. Protection was determined if ASDs significantly increased the temperature at which Scn1aA1783V/WT mice seized. ResultsApproximately 50% of Scn1aA1783V/WT survive to adulthood and all have hyperthermia-induce seizures. The results suggest that hyperthermia-induced seizures in this model of DS are highly refractory to a battery of ASDs. Exceptions were clobazam, tiagabine, and the combination of clobazam and valproic acid with add-on stiripentol, which elevated seizure thresholds SignificanceOverall, the data demonstrate the proposed model for DS is suitable for screening novel compounds for the ability to block hyperthermia-induced seizures and heterozygous mice can be evaluated repeatedly over the course of several weeks, allowing for higher throughput screening. Key Points O_LIScn1aA1783V/WT mice have a 50% survival rate and all have hyperthermia-induced seizures. C_LIO_LICommon DS treatments such as CLB and combinatorial therapy of CLB, VPA, and STP increase temperature thresholds in Scn1aA1783V/WT mice. C_LIO_LISodium channel blockers, such as CBZ and LTG, decrease temperature thresholds of Scn1aA1783V/WT mice as predicted. C_LIO_LIScn1aA1783V/WT mice are highly pharmacoresitant to common ASDs C_LIO_LIThe Scn1aA1783V/WT may be a useful preclinical drug screening platform for the treatment of DS. C_LI

pharmacology and toxicology↗

bioRxiv2020

Kinome Profiling of Gastrointestinal Stromal Tumors Using Multiplexed Inhibitor Beads and Mass Spectrometry Identifies Wee1 as Candidate Therapeutic Target

Management of gastrointestinal stromal tumor (GIST) has been revolutionized by the identification of activating mutations in KIT and PDGFRA, and the clinical application of receptor tyrosine kinase (RTK) inhibitors in the advanced disease setting. Stratification of GIST into molecularly defined subsets provides insight into clinical behavior and response to approved targeted therapies. Although these RTK inhibitors are effective in the majority of GIST, resistance to these agents remains a significant clinical problem. Development of effective treatment strategies for refractory GIST subtypes requires identification of novel targets to provide additional therapeutic options. Global kinome profiling has the potential to identify critical signaling networks and reveal protein kinases that are essential in GIST. Using Multiplexed Inhibitor Beads and Mass Spectrometry, we explored the majority of the kinome in GIST specimens from the three most common molecular subtypes to identify novel kinase targets. Kinome profiling revealed distinct signatures in GIST subtypes and identified kinases that are universally activated in all GIST, as well as kinases that are unique to each subtype. Kinome profiling in combination with loss-of-function assays identified a significant role for the G2-M tyrosine kinase, Wee1, in GIST cell survival. In vitro and in vivo studies revealed significant efficacy of MK-1775 (Wee1 inhibitor) in combination with avapritinib in KIT and PDGFRA-mutant GIST cell lines, and notable efficacy of MK-1775 as a single agent in the PDGFRA-mutant line. These studies provide strong preclinical justification for the use of MK-1775 in GIST.

cancer biology↗

bioRxiv2020

Kinome Profiling of Primary Endometrial Tumors Using Multiplexed Inhibitor Beads and Mass Spectrometry Identifies SRPK1 As Candidate Therapeutic Target

Protein kinases (collectively, termed the kinome) represent one of the most tractable drug targets in the pursuit of new and effective cancer treatments. However, less than 20% of the kinome is currently being explored as primary targets for cancer therapy, leaving the majority of the kinome untargeted for drug therapy. Chemical proteomics approaches such as Multiplexed Inhibitor Beads and Mass Spectrometry (MIB-MS) have been developed that measure the abundance of a significant portion of the kinome, providing a strategy to interrogate kinome landscapes and dynamics. Kinome profiling of cancer cell lines using MIB-MS has been extensively characterized, however, application of this method to measure tissue kinome(s) has not been thoroughly explored. Here, we present a quantitative proteomics workflow specifically designed for kinome profiling of tissues that pairs MIB-MS with a newly designed super-SILAC kinome standard. Using this workflow, we mapped the kinome landscape of endometrial carcinoma (EC) tumors and normal endometrial (NE) tissues and identified several kinases overexpressed in EC tumors, including Serine/Arginine-Rich Splicing Factor kinase, (SRPK1). Immunohistochemical (IHC) analysis of EC tumor TMAs confirmed MIB-MS findings and showed SRPK1 protein levels were highly expressed in endometrioid and uterine serous cancer (USC) histological subtypes. Querying large-scale genomics studies of EC tumors revealed high expression of SRPK1 correlated with poor survival. Inhibition of SRPK1 in USC cells altered mRNA splicing, downregulating several oncogenes including MYC and Survivin resulting in apoptosis. Taken together, we present a SILAC-based MIB-MS kinome profiling platform for measuring kinase abundance in tumor tissues, and demonstrate its application to identify SRPK1 as a plausible kinase drug target for the treatment of EC.

cancer biology↗