Biology subjects

Schafer, R.

Publications and source records attributed to Schafer, R..

8 recordsLinked to original sources

bioRxiv2025

PANK2-mediated de novo CoA synthesis is required for metabolic switching to fatty acid oxidation

Humans have 3 different PANK enzymes (PANK1-3) that catalyze the first step in the de novo synthesis of Coenzyme A (CoA). All PANKs are feedback inhibited by acyl-CoAs but only PANK2 can overcome this inhibition by binding palmitoyl-carnitine. Previous studies, conducted under glucose-replete conditions, have failed to detect a PANK2-mediated contribution to CoA synthesis. We found that exposure to BSA-conjugated palmitate (PAL-BSA) led to activation of fatty acid oxidation (FAO) and the accumulation of both palmitoyl-carnitine and palmitoyl-CoA in HEK293T cells, suggesting that PANK2 is active under these conditions. Isotope tracing experiments with 13C15N-pantothenate showed that PANK2 uniquely sustains de novo CoA synthesis and high production of Acetyl-CoA in the presence of long-chain fatty acids, indicating that FAO is limited by CoA availability in these conditions. Consistent with this mechanism, fibroblasts from PKAN patients exhibited impaired oxidation of palmitoyl-carnitine, confirming the functional relevance of our results in a disease context.

biochemistry↗

bioRxiv2025

Contribution of S1pr1-featured astrocyte subpopulation to cisplatin-induced neuropathic pain

Chemotherapy-induced peripheral neuropathy accompanied by neuropathic pain (CIPN) is a major neurotoxicity of cisplatin, a platinum-based drug widely used for lung, ovarian, and testicular cancer treatment. CIPN causes drug discontinuation and severely impacts life quality with no FDA-approved interventions. We previously reported that platinum-based drugs increase levels of sphingosine 1-phosphate (S1P) in the spinal cord and drive CIPN through activating the S1P receptor subtype 1 (S1PR1). However, the mechanisms engaged downstream of S1PR1 remain poorly understood. Using single cell transcriptomics on male mouse spinal cord, our findings uncovered subpopulation-specific responses to cisplatin associated with CIPN. Particularly, cisplatin increased the proportion of astrocytes with high expression levels of S1pr1 (S1pr1high astrocytes), specific to which a Wnt signaling pathway was identified. To this end, several genes involved in Wnt signaling, such as the fibroblast growth factor receptor 3 gene (Fgfr3), were highly expressed in S1pr1high astrocytes. The functional S1PR1 antagonist, ozanimod, prevented cisplatin-induced neuropathic pain and astrocytic upregulation of the Wnt signaling pathway genes. FGFR3 belongs to the FGF/FGFR family which often signals to activate Wnt signaling. Intrathecal injection of the FGFR3 antagonist, PD173074, prevented the development of CIPN in male mice. These data not only highlight FGFR3 as one of the astrocytic targets of S1PR1 but raise the possibility that S1PR1-induced engagement of Wnt signaling in S1pr1high astrocytes may contribute to CIPN. Overall, our results provide a comprehensive mapping of cellular and molecular changes engaged in cisplatin-induced neuropathic pain and decipher novel S1PR1-based mechanisms of action.

neuroscience↗

bioRxiv2024

Neural mechanisms underlying robust target selection in response to microstimulation of the oculomotor system

Despite its prevalence in studying the causal roles of different brain circuits in cognitive processes, electrical microstimulation often results in inconsistent behavioral effects. These inconsistencies are assumed to be due to multiple mechanisms, including habituation, compensation by other brain circuits, and contralateral suppression. Considering the presence of reinforcement in most experimental paradigms, we hypothesized that interactions between reward feedback and microstimulation could contribute to inconsistencies in behavioral effects of microstimulation. To test this, we analyzed data from electrical microstimulation of the frontal eye field of male macaques during a value-based decision-making task and constructed network models to capture choice behavior. We found evidence for microstimulation-dependent adaptation in saccadic choice, such that in stimulated trials, monkeys choices were biased toward the target in the response field of the microstimulated site (Tin). In contrast, monkeys showed a bias away from Tin in non-stimulated trials following microstimulation. Critically, this bias slowly decreased as a function of the time since the last stimulation. Moreover, microstimulation-dependent adaptation was influenced by reward outcomes in preceding trials. Despite these local effects, we found no evidence for the global effects of microstimulation on learning and sensitivity to the reward schedule. By simulating choice behavior across various network models, we found a model in which microstimulation and reward-value signals interact competitively through reward-dependent plasticity can best account for our observations. Our findings indicate a reward-dependent compensatory mechanism that enhances robustness to perturbations within the oculomotor system and could explain the inconsistent outcomes observed in previous microstimulation studies.

neuroscience↗

bioRxiv2024

eIF2α phosphorylation evokes dystonia-like movements with D2-receptor and cholinergic origin and abnormal neuronal connectivity

Dystonia is the 3rd most common movement disorder. Dystonia is acquired through either injury or genetic mutations, with poorly understood molecular and cellular mechanisms. Eukaryotic initiation factor alpha (eIF2) controls cell state including neuronal plasticity via protein translation control and expression of ATF4. Dysregulated eIF2 phosphorylation (eIF2-P) occurs in dystonia patients and models including DYT1, but the consequences are unknown. We increased/decreased eIF2-P and tested motor control and neuronal properties in a Drosophila model. Bidirectionally altering eIF2-P produced dystonia-like abnormal posturing and dyskinetic movements in flies. These movements were also observed with expression of the DYT1 risk allele. We identified cholinergic and D2-receptor neuroanatomical origins of these dyskinetic movements caused by genetic manipulations to dystonia molecular candidates eIF2-P, ATF4, or DYT1, with evidence for decreased cholinergic release. In vivo, increased and decreased eIF2-P increase synaptic connectivity at the NMJ with increased terminal size and bouton synaptic release sites. Long-term treatment of elevated eIF2-P with ISRIB restored adult longevity, but not performance in a motor assay. Disrupted eIF2-P signaling may alter neuronal connectivity, change synaptic release, and drive motor circuit changes in dystonia.

neuroscience↗

bioRxiv2021

The WRAD complex represents a therapeutically exploitable target for cancer stem cells in glioblastoma

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors that are driven by populations of cancer stem cells (CSCs). Despite their importance for tumor growth, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1-family-mediated Lys4 methylation of histone H3, associated with positive regulation of transcription. In GBM CSC models, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. We incorporated a SOX2/OCT4 motif driven GFP reporter system into our CSC cell models and found that WDR5 inhibitor treatment diminished reporter activity. Further, WDR5 inhibitor treatment altered the stem cell state, disrupting CSC in vitro growth and self-renewal as well as in vivo tumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers. SignificanceIn this study, we perform an epigenetic-focused functional genomics screen in glioblastoma organoids and identify WDR5 as an essential epigenetic regulator in the SOX2-enriched, therapy resistant cancer stem cell niche. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=135 HEIGHT=200 SRC="FIGDIR/small/461125v2_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@bc5c5eorg.highwire.dtl.DTLVardef@545a2borg.highwire.dtl.DTLVardef@1ebd3c9org.highwire.dtl.DTLVardef@19d5d30_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

Identification of a Nervous System Gene Expression Signature in Colon Cancer Stem Cells Reveals a Role for Neural Crest Regulators EGR2 and SOX2 in Tumorigenesis

Recent data support a hierarchical model of colon cancer driven by a population of cancer stem cells (CSCs). Greater understanding of the mechanisms that regulate CSCs may therefore lead to more effective treatments. Serial limiting dilution xenotransplantation assays of colon cancer patient-derived tumors demonstrated ALDHPositive cells to be enriched for tumorigenic self-renewing CSCs. In order to identify CSC modulators, we performed RNA-sequencing analysis of ALDHPositive CSCs from a panel of colon cancer patient-derived organoids (PDOs) and xenografts (PDXs). These studies demonstrated CSCs to be enriched for embryonic and neural development gene sets. Functional analyses of genes differentially expressed in both ALDHPositive PDO and PDX CSCs demonstrated the neural crest stem cell (NCSC) regulator and wound response gene EGR2 to be required for CSC tumorigenicity and to control expression of homeobox superfamily embryonic master transcriptional regulator HOX genes and the embryonic and neural stem cell regulator SOX2. In addition, we identify EGR2, HOXA2, HOXA4, HOXA5, HOXA7, HOXB2, HOXB3 and the tumor suppressor ATOH1 as new prognostic biomarkers in colorectal cancer.

cell biology↗

bioRxiv2021

RNA-Sequencing of Long-Term Label-Retaining Colon Cancer Stem Cells Identifies Novel Regulators of Quiescence

Recent data suggests that colon tumors contain a subpopulation of therapy resistant quiescent cancer stem cells (qCSCs) that are the source of relapse following treatment. Here, using colon cancer patient-derived organoids (PDOs) and xenograft (PDX) models, we identify a rare population of long-term label-retaining (PKH26Positive) qCSCs that can re-enter the cell cycle to generate new tumors. RNA-sequencing analyses demonstrated that these cells are enriched for stem cell associated gene sets such as Wnt and hedgehog signaling, epithelial-to-mesenchymal transition (EMT), embryonic development, tissue development and p53 pathway but have downregulated expression of genes associated with cell cycle, transcription, biosynthesis and metabolism. Furthermore, qCSCs are enriched for p53 interacting negative regulators of cell cycle, including AKAP12, CD82, CDKN1A, FHL2, GPX3, KIAA0247, LCN2, TFF2, UNC5B and ZMAT3, that we show are indicators of poor prognosis and may be targeted for qCSC abolition. Interestingly, CD82, KIAA0247 and UNC5B proteins localize to the cell surface and may therefore be potential markers for the prospective isolation of qCSCs. These data support the temporal inhibition of p53 signaling for the elimination of qCSCs and prevention of relapse in colorectal cancer.

cell biology↗

bioRxiv2020

Experimental Stroke Induces Chronic Gut Dysbiosis and Neuroinflammation in Male Mice

Recent literature implicates gut epithelia mucosa and intestinal microbiota as important players in post-stroke morbidity and mortality. As most studies have focused on the acute effects of stroke on gut dysbiosis, our study objective was to measure chronic, longitudinal changes in the gut microbiota and intestinal pathology following ischemic stroke. We hypothesized that mice with experimental ischemic stroke would exhibit chronic gut dysbiosis and intestinal pathology up to 36 days post-stroke compared to sham controls. Male C57BL/6J mice were subjected to 60 minutes of transient middle cerebral artery occlusion (tMCAO) or sham surgery. To determine the long-term effects of tMCAO on gut dysbiosis, fecal boli were collected pre- and post-tMCAO on days 0, 3, 14, and 28. Bioinformatics analysis demonstrate significant differences in abundance among Firmicutes and Bacteroidetes taxa at the phylum, family, and species levels in tMCAO compared to sham mice that persisted up to one month post-stroke. The most persistent changes in post-stroke microbial abundance were a decrease in bacteria family S24-7 and significant increases in Ruminococcaceae. Overall, these changes resulted in a persistently increased Firmicutes:Bacteroidetes ratio in stroke animals. Intestinal histopathology showed evidence of chronic intestinal inflammation that included marked increases in immune cell infiltration with mild-moderate epithelial hyperplasia and villous blunting. Increased astrocyte and microglial activity were also detected one-month post-stroke. These results demonstrate that acute, post-stroke disruption of the gut-brain-microbiota axis progresses to chronic gut dysbiosis, intestinal inflammation, and chronic neuroinflammation. Clinical PerspectivesO_LIThe microbiota-gut-brain axis, recently implicated in several neurological disorders, remains largely unexplored at chronic time points post-tMCAO. C_LIO_LIOur results demonstrate chronic gut dysbiosis, prolonged behavioral deficits, and persistent cerebral and intestinal inflammation post-tMCAO in male C57BL/6J mice. C_LIO_LIThese results suggest that manipulation of microbiota may help reduce poor outcomes after stroke and lead to improved post-stroke functional recovery. C_LI

neuroscience↗