Biology subjects

Knudsen, A.

Publications and source records attributed to Knudsen, A..

5 recordsLinked to original sources

bioRxiv2026

A synthetic PGC1α co-activator reprograms microglial immunometabolism to preserve cone function in retinal degeneration

Retinitis pigmentosa (RP) is genetically heterogeneous, yet initiating mutations converge on primary rod degeneration followed by cone dysfunction and loss. Activated microglia are implicated in this secondary phase, but whether selective immunometabolic reprogramming of microglia preserves retinal function remains unknown. In human iPSC-derived microglia and retinal CD11b cells, PGC1 induction suppressed LPS-driven interferon signaling and cytokine release, yet Seahorse analysis and isotopologue tracing showed that energy metabolism remained altered. We therefore fused PGC1 to a multivalent p65-HSF1 module, yielding ~2.5-fold higher nuclear receptor output than native PGC1 and coordinated engagement of oxidative and anti-inflammatory programs in mouse retinal CD11b cells. Microglia-restricted expression in humanized RHOP347L mice preserved photopic responses through six months, increased retinal thickness and promoted ramified subretinal IBA1 morphology. Microglial immunometabolism thus represents a potentially genotype-independent neuroprotective target in RP, and engineered PGC1 co-activators provide a means to sustain oxidative metabolism under inflammatory pressure.

immunology↗

bioRxiv2021

Biophysical demonstration of co-packaging of glutamate and GABA in individual synaptic vesicles in the central nervous system

Many mammalian neurons release multiple neurotransmitters to activate diverse classes of ionotropic receptors on their postsynaptic targets. Entopeduncular nucleus somatostatin (EP Sst+) neurons that project to the lateral habenula (LHb) release both glutamate and GABA, but it is unclear if these are packaged into the same or segregated pools of synaptic vesicles. Here we describe a novel method combining electrophysiology, spatially-patterned optogenetics, and computational modeling designed to analyze the mechanism of glutamate/GABA corelease. We find that the properties of PSCs elicited in LHb neurons by optogenetic activation of EP Sst+ terminals are only consistent with co-packaging of glutamate and GABA into individual vesicles. Furthermore, serotonin, which acts presynaptically to weaken EP Sst+ to LHb synapses, does so by altering the release probability of vesicles containing both transmitters. Our approach is broadly applicable to the study of multi-transmitter neurons throughout the brain and our results constrain mechanisms of neuromodulation in LHb.

neuroscience↗

bioRxiv2020

Altered lipid metabolism marks glioblastoma stem and non-stem cells in separate tumor niches

BackgroundGlioblastoma (GBM) is marked by cellular heterogeneity, including metabolic heterogeneity, that varies among cellular microenvironments in the same tumor. Altered cellular metabolism in cancer is well-established, but how lipid metabolism is altered to suit different microenvironmental conditions and cellular states within a tumor remains unexplored. MethodsWe assessed GBM organoid models that mimic the transition zone between nutrient-rich and nutrient-poor pseudopalisading/perinecrotic tumor zones and performed spatial RNA-sequencing of cells to interrogate lipid metabolism. Using targeted lipidomic analysis, we assessed differences in acutely enriched cancer stem cells (CSCs) and non-CSCs from multiple patient-derived models to explore the link between the stem cell state and lipid metabolism. ResultsSpatial analysis revealed a striking difference in lipid content between microenvironments, with lipid enrichment in the hypoxic organoid cores and the perinecrotic and pseudopalisading regions of primary patient tumors. This was accompanied by regionally restricted upregulation of hypoxia-inducible lipid droplet-associated (HILPDA) gene expression in organoid cores and in clinical GBM specimens, but not lower-grade brain tumors, that was specifically localized to pseudopalisading regions of patient tumors. CSCs have low lipid droplet accumulation compared to non-CSCs in organoid models and xenograft tumors, and prospectively sorted lipid-low GBM cells are functionally enriched for stem cell activity. Targeted lipidomic analysis revealed that CSCs had decreased levels of major classes of neutral lipids compared to non-CSCs but had significantly increased polyunsaturated fatty acid production due to high fatty acid desaturase (FADS1/2) expression. ConclusionsOur data demonstrate that lipid metabolism is differentially altered across GBM microenvironments and cellular hierarchies, providing guidance for targeting of these altered lipid metabolic pathways. Key pointsO_LIGBM cells in nutrient-poor tumor regions have increased accumulation of lipid droplets. C_LIO_LICSCs have reduced lipid content compared to non-CSCs. C_LIO_LIGBM CSCs and non-CSCs have disparate lipid metabolisms that may be uniquely targetable. C_LI Importance of the StudyMetabolic targeting has long been advocated as a therapy against many tumors including GBM, and it remains an outstanding question whether cancer stem cells (CSCs) have altered lipid metabolism. We demonstrated striking differences in lipid metabolism between diverse cell populations from the same patient. These spatially and phenotypically distinct lipid phenotypes occur clinically in the majority of patients and can be recapitulated in laboratory models. Lipidomic analysis of multiple patient-derived models shows a significant shift in lipid metabolism between GBM CSCs and non-CSCs, suggesting that lipid levels may not be simply a product of the microenvironment but also may be a reflection of cellular state. Our results suggest that therapeutic targeting of GBM lipid metabolism must consider multiple separate tumor cell populations to be effective, and we provide a methodologic framework for studying these metabolically diverse cellular populations.

cancer biology↗

bioRxiv2020

Identification of SARS-CoV2-mediated suppression of NRF2 signaling reveals a potent antiviral and anti-inflammatory activity of 4-octyl-itaconate and dimethyl fumarate

Antiviral strategies to inhibit Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and the pathogenic consequences of COVID-19 are urgently required. Here we demonstrate that the NRF2 anti-oxidant gene expression pathway is suppressed in biopsies obtained from COVID-19 patients. Further, we uncover that NRF2 agonists 4-octyl-itaconate (4-OI) and the clinically approved dimethyl fumarate (DMF) induce a cellular anti-viral program, which potently inhibits replication of SARS-CoV2 across cell lines. The anti-viral program extended to inhibit the replication of several other pathogenic viruses including Herpes Simplex Virus-1 and-2, Vaccinia virus, and Zika virus through a type I interferon (IFN)-independent mechanism. In addition, induction of NRF2 by 4-OI and DMF limited host inflammatory responses to SARS-CoV2 infection associated with airway COVID-19 pathology. In conclusion, NRF2 agonists 4-OI and DMF induce a distinct IFN-independent antiviral program that is broadly effective in limiting virus replication and suppressing the pro-inflammatory responses of human pathogenic viruses, including SARS-CoV2. One Sentence SummaryNRF2 agonists 4-octyl-itaconate (4-OI) and dimethyl fumarate inhibited SARS-CoV2 replication and virus-induced inflammatory responses, as well as replication of other human pathogenic viruses.

microbiology↗

bioRxiv2018

Immunostimulatory guide RNAs mediate potent antiviral response

Genome-editing with CRISPR has emerged as a technology with broad therapeutic potential. However, it is unclear whether CRISPR will elicit innate immune responses, which could impact both positively and negatively on the desired therapeutic effects. Here, we have examined the immune-stimulatory properties of different variants of guide RNAs (gRNAs) - in vitro transcribed gRNA (IVT-gRNA) and synthetic gRNAs with or without chemical modifications, full-length or duplexed. We find that only IVT-gRNA evokes strong expression of cytokines in a panel of cell lines while all the synthetic RNAs do not. We further find that sensing of IVT-gRNA proceeds mainly through the RIG-I/MAVS RNA sensing axis. One potential use of CRISPR is for antiviral therapy. The antiviral actions of the gRNA tested up until now have been relying purely on the gene editing function of the CRISPR machinery, which weakens its feasibility due to the difficulty to target all infected cells. When IVT-gRNA was combined with unmodified Cas9 mRNA, which also induces cytokine expression, strong immune response was obtained while maintaining nuclease activity of CRISPR. Remarkably, such combination inhibited herpes simplex virus type-1 (HSV-1) replication even though the nuclease activity was modest, and provided bystander protection to the cells that were not transfected with CRIPSR molecules. The antiviral activity of IVT-gRNA was also observed in vivo in HSV-1-infected Cas9+ mice, thus demonstrating the therapeutic potential. Our study further extends the applications of CRISPR by exploiting the immunostimulatory function of gRNAs.

immunology↗