Biology subjects

Merson, T. D.

Publications and source records attributed to Merson, T. D..

4 recordsLinked to original sources

bioRxiv2024

HB-EGF and EGF infusion following CNS demyelination mitigates age-related decline in regeneration of oligodendrocytes from neural precursor cells originating in the ventricular-subventricular zone

In multiple sclerosis (MS), chronic demyelination initiated by immune-mediated destruction of myelin, leads to axonal damage and neuronal cell death, resulting in a progressive decline in neurological function. The development of interventions that potentiate remyelination could hold promise as a novel treatment strategy for MS. To this end, our group has demonstrated that neural precursor cells (NPCs) residing in the ventricular-subventricular zone (V-SVZ) of the adult mouse brain contribute significantly to remyelination in response to central nervous system (CNS) demyelination and can regenerate myelin of normal thickness. However, aging takes its toll on the regenerative potential of NPCs and reduces their contribution to remyelination. In this study, we investigated how aging influences the contribution of NPCs to oligodendrogenesis during the remyelination process and whether the delivery of growth factors into the brains of aged mice could potentiate the oligodendrogenic potential of NPCs. To enable us to map the fate of NPCs in response to demyelination induced at different postnatal ages, Nestin-CreERT2; Rosa26-LSL-eYFP mice were gavaged with tamoxifen at either 8 weeks, 30 weeks or one year of age before being challenged with cuprizone for a period of six weeks. Using osmotic minipumps, we infused heparin-binding EGF-like growth factor (HB-EGF), and/or epidermal growth factor (EGF) into the cisterna magna for a period of two weeks beginning at the peak of cuprizone-induced demyelination (n=6-8 mice per group). Control mice received artificial cerebrospinal fluid (vehicle) alone. Mice were perfused six weeks after cuprizone withdrawal and the contribution of NPCs to oligodendrocyte regeneration in the corpus callosum was assessed. Our data reveal that although NPC-derived oligodendrocyte generation declined dramatically with age, this decline was partially reversed by growth factor infusion. Notably, co-infusion of EGF and HB-EGF increased oligodendrocyte regeneration twofold in some regions of the corpus callosum. Our results shed light on the beneficial effects of EGF and HB-EGF for increasing the contribution of NPCs to remyelination and indicate their therapeutic potential to combat the negative effects of aging upon remyelination efficacy.

neuroscience↗

bioRxiv2022

5' transgenes drive leaky expression of 3' transgenes in inducible bicistronic vectors

Molecular cloning techniques enabling contemporaneous expression of two or more protein-coding sequences in a cell type of interest provide an invaluable tool for understanding the molecular regulation of cellular functions. DNA recombination employing the Cre-lox system is commonly used as a molecular switch for inducing the expression of recombinant proteins encoded within a bicistronic cassette. In such an approach, the two protein-coding sequences are separated by a 2A peptide or internal ribosome entry site (IRES), and expression is designed to be strictly Cre-dependent by using a lox-STOP-lox cassette or flip-excision (FLEX) switch. However, low-level or leaky expression of recombinant proteins is often observed in the absence of Cre activity, potentially compromising the utility of this approach. To investigate the mechanism of leaky gene expression, we generated pCAG-lox-GFP-STOP-lox-Transgene A-2A-Transgene B vectors, which are designed to express nuclear-targeted GFP in the absence of Cre, and express both transgenes A and B after Cre-mediated recombination. We found that cells transfected with these bicistronic vectors exhibited low-level Cre-independent expression specifically of the transgene positioned 3' of the 2A peptide. We observed similar results in vivo by viral transduction of the adult mouse cerebral cortex with AAV-mutagenesis of putative transcription factor binding sites that the 5' transgene confers promoter-like activity that drives expression of the 3' transgene. Finally, we demonstrate that inclusion of an additional lox-STOP-lox cassette between the 2A sequence and 3' transgene dramatically reduces the extent of Cre-independent leaky gene expression. Our findings highlight that caution should be applied to the use of Cre-dependent bicistronic constructs when tight regulation of transgene expression is desired and provide a guide to preventing leaky gene expression when the expression of more than one protein is required.

molecular biology↗

bioRxiv2021

A Novel Pharmacogenetic Model for Highly Efficient Ablation of Oligodendrocyte Progenitor Cells in the Adult Mouse CNS

Approaches to investigate adult oligodendrocyte progenitor cells (OPCs) by targeted cell ablation in the rodent central nervous system have been limited by methodological challenges resulting in only partial and transient OPC depletion. We have developed a novel pharmacogenetic model of conditional OPC ablation, eliminating 98.6% of all OPCs throughout the brain. By combining recombinase-based transgenic and viral strategies for targeting OPCs and ventricular-subventricular zone (V-SVZ)-derived neural precursor cells (NPCs), we found new PDGFRA-expressing cells born in the V-SVZ repopulated the OPC-deficient brain starting 12 days after OPC ablation. Our data reveal that OPC depletion induces V-SVZ-derived NPCs to generate vast numbers of PDGFRA+ NG2+ cells with the capacity to migrate and proliferate extensively throughout the dorsal anterior forebrain. Further application of this novel approach to ablate OPCs will advance knowledge of the function of both OPCs and oligodendrogenic NPCs in health and disease.

neuroscience↗

bioRxiv2020

Primate-specific response of astrocytes to stroke limits peripheral macrophage infiltration

Reactive astrocytes play critical roles after brain injuries but their precise function in stroke is not well defined. Here, we utilized single nuclei transcriptomics to characterize astrocytes after ischemic stroke in nonhuman primate (NHP) marmoset monkey primary visual cortex. We identified 19 putative subtypes of astrocytes from injured and uninjured brain hemispheres and observed nearly complete segregation between stroke and control astrocyte clusters. We then screened for genes that might be limiting stroke recovery and discovered that one neurite-outgrowth inhibitory protein, NogoA, previously associated with oligodendrocytes but not astrocytes, was expressed in numerous reactive astrocyte subtypes. NogoA upregulation on reactive astrocytes was confirmed in vivo for NHP and human, but not observed to the same extent in rodent. Further in vivo and in vitro studies determined that NogoA mediated an anti-inflammatory response which limits deeper infiltration of peripheral macrophages from the lesion during the subacute post-stroke period. Specifically, these findings are relevant to the development of NogoA-targeting therapies shortly after ischemic stroke. Our findings have uncovered the complexity and species specificity of astrocyte responses, which need to be considered more when investigating novel therapeutics for brain injury.

neuroscience↗